Inhibitors of interleukin-1β converting enzyme

ABSTRACT

The present invention relates to novel classes of compounds which are inhibitors of interleukin-1β converting enzyme (“ICE”). This invention also relates to pharmaceutical compositions comprising these compounds. The compounds and pharmaceutical compositions of this invention are particularly well suited for inhibiting ICE activity and consequently, may be advantageously used as agents against interleukin-1-(“IL-1”), apoptosis-, interferon-γ inducing factor-(IGIF), interferon-γ-(“IFN-γ”) mediated diseases, excess dietary alcohol intake diseases, or viral diseases, including inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, and degenerative diseases. This invention also relates to methods for inhibiting ICE activity and decreasing IGIF production and IFN-γ production and methods for treating interleukin-1, apoptosis- and interferon-γ-mediated diseases using the compounds and compositions of this invention. This invention also relates to methods of preparing the compounds of this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/326,495,filed Jun. 4, 1999, now U.S. Pat. No. 6,329,365, which is a continuationof PCT/US97/22289 filed Dec. 5, 1997, which claims benefit of U.S.provisional patent application No. 60/053,001, filed Jun. 26, 1997, andwhich claims benefit of U.S. provisional patent application No.60/042,660, filed Apr. 4, 1997, and which claims benefit of U.S.provisional patent application No. 60/032,792, filed Dec. 6, 1996.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel classes of compounds which areinhibitors of interleukin-1β converting enzyme (“ICE”). This inventionalso relates to pharmaceutical compositions comprising these compounds.The compounds and pharmaceutical compositions of this invention areparticularly well suited for inhibiting ICE activity and consequently,may be advantageously used as agents against interleukin-1-(“IL-1”),apoptosis-, interferon-γ inducing factor-(IGIF), interferon-γ-(“IFN-γ”)mediated diseases, excess dietary alcohol intake diseases, or viraldiseases, including inflammatory diseases, autoimmune diseases,destructive bone disorders, proliferative disorders, infectiousdiseases, and degenerative diseases. This invention also relates tomethods for inhibiting ICE activity and decreasing IGIF production andIFN-γ production and methods for treating interleukin-1, apoptosis- andinterferon-γ-mediated diseases using the compounds and compositions ofthis invention. This invention also relates to methods of preparing thecompounds of this invention.

BACKGROUND OF THE INVENTION

Interleukin 1 (“IL-1”) is a major pro-inflammatory and immunoregulatoryprotein that stimulates fibroblast differentiation and proliferation,the production of prostaglandins, collagenase and phospholipase bysynovial cells and chondrocytes, basophil and eosinophil degranulationand neutrophil activation. Oppenheim, J. H. et al., Immunology Today, 7,pp. 45-56 (1986). As such, it is involved in the pathogenesis of chronicand acute inflammatory and autoimmune diseases. For example, inrheumatoid arthritis, IL-1 is both a mediator of inflammatory symptomsand of the destruction of the cartilage proteoglycan in afflictedjoints. Wood, D. D. et al., Arthritis Rheum. 26, 975, (1983); Pettipher,E. J. et al., Proc. Natl. Acad. Sci. U.S.A. 71, 295 (1986); Arend, W. P.and Dayer, J. M., Arthritis Rheum. 38, 151 (1995). IL-1 is also a highlypotent bone resorption agent. Jandiski, J. J., J. Oral Path 17, 145(1988); Dewhirst, F. E. et al., J. Immunol. 8, 2562 1985). It isalternately referred to as “osteoclast activating factor” in destructivebone diseases such as osteoarthritis and multiple myeloma. Bataille, R.et al., Int. J. Clin. Lab. Res. 21(4), 283 (1992). In certainproliferative disorders, such as acute myelogenous leukemia and multiplemyeloma, IL-1 can promote tumor cell growth and adhesion. Bani, M. R.,J. Natl. Cancer Inst. 83, 123 (1991); Vidal-Vanaclocha, F., Cancer Res.54, 2667 (1994). In these disorders, IL-1 also stimulates production ofother cytokines such as IL-6, which can modulate tumor development(Tartour et al., Cancer Res. 54, 6243 (1994). IL-1 is predominantlyproduced by peripheral blood monocytes as part of the inflammatoryresponse and exists in two distinct agonist forms, IL-1α and IL-1β.Mosely, B. S. et al., Proc. Nat. Acad. Sci., 84, pp.4572-4576 (1987);Lonnemann, G. et al., Eur.J. Immunol., 19, pp.1531-1536 (1989).

IL-1β is synthesized as a biologically inactive precursor, pIL-1β.pIL-1β lacks a conventional leader sequence and is not processed by asignal peptidase. March, C. J., Nature, 315, pp.641-647 (1985). Instead,pIL-1β is cleaved by interleukin-1β converting enzyme (“ICE”) betweenAsp-116 and Ala-117 to produce the biologically active C-terminalfragment found in human serum and synovial fluid. Sleath, P. R., et al.,J. Biol. Chem., 265, pp.14526-14528 (1992); A. D. Howard et al., J.Immunol., 147, pp.2964-2969 (1991). ICE is a cysteine protease localizedprimarily in monocytes. It converts precursor IL-1β to the mature form.Black, R. A. et al., FEBS Lett., 247, pp.386-390 (1989); Kostura, M. J.et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp.5227-5231 (1989).Processing by ICE is also necessary for the transport of mature IL-1βthrough the cell membrane.

ICE, or its homologs, also appears to be involved in the regulation ofprogrammed cell death or apoptosis. Yuan, J. et al., Cell, 75,pp.641-652 (1993); Miura, M. et al., Cell, 75, pp.653-660 (1993);Nett-Fiordalisi, M. A. et al., J. Cell Biochem., 17B, p.117 (1993). Inparticular, ICE or ICE homologs are thought to be associated with theregulation of apoptosis in neurodegenerative diseases, such asAlzheimer's and Parkinson's disease. Marx,J. and M. Baringa, Science,259, pp.760-762 (1993); Gagliardini, V. et al., Science, 263, pp.826-828(1994). Therapeutic applications for inhibition of apoptosis may includetreatment of Alzheimer's disease, Parkinson's disease, stroke,myocardial infarction, spinal atrophy, and aging.

ICE has been demonstrated to mediate apoptosis (programmed cell death)in certain tissue types. Steller, H., Science, 267, p. 1445 (1995);Whyte, M. and Evan, G., Nature, 376, p. 17 (1995); Martin, S. J. andGreen, D. R., Cell, 82, p. 349 (1995); Alnemri, E. S., et al., J. Biol.Chem., 270, p. 4312 (1995); Yuan, J. Curr. Opin. Cell Biol., 7, p. 211(1995). A transgenic mouse with a disruption of the ICE gene isdeficient in Fas-mediated apoptosis (Kuida, K. et al., Science 267, 2000(1995)). This activity of ICE is distinct from its role as theprocessing enzyme for pro-IL1β. It is conceivable that in certain tissuetypes, inhibition of ICE may not affect secretion of mature IL-1β, butmay inhibit apoptosis.

Enzymatically active ICE has been previously described as a heterodimercomposed of two subunits, p20 and p10 (20 kDa and 10 kDa molecularweight, respectively). These subunits are derived from a 45 kDaproenzyme (p45) by way of a p30 form, through an activation mechanismthat is autocatalytic. Thornberry, N. A. et al., Nature, 356, pp.768-774(1992). The ICE proenzyme has been divided into several functionaldomains: a prodomain (p14), a p22/20 subunit, a polypeptide linker and ap10 subunit. Thornberry et al., supra; Casano et al., Genomics, 20, pp.474-481 (1994).

Full length p45 has been characterized by its cDNA and amino acidsequences. PCT patent applications WO 91/15577 and WO 94/00154. The p20and p10 cDNA and amino acid sequences are also known. Thornberry et al.,supra. Murine and rat ICE have also been sequenced and cloned. They havehigh amino acid and nucleic acid sequence homology to human ICE. Miller,D. K. et al., Ann. N.Y. Acad. Sci., 696, pp. 133-148 (1993); Molineaux,S. M. et al., Proc. Nat. Acad. Sci., 90, pp. 1809-1813 (1993). Thethree-dimensional structure of ICE has been determined at atomicresolution by X-ray crystallography. Wilson, K. P., et al., Nature, 370,pp. 270-275 (1994). The active enzyme exists as a tetramer of two p20and two p10 subunits.

Additionally, there exist human homologs of ICE with sequencesimilarities in the active site regions of the enzymes. Such homologsinclude TX (or ICE_(rel-II) or ICH-2) (Faucheu, et al., EMBO J., 14, p.1914 (1995); Kamens J., et al., J. Biol. Chem., 270, p. 15250 (1995);Nicholson et al., J. Biol. Chem., 270 15870 (1995)), TY (orICE_(rel-III)) (Nicholson et al., J. Biol. Chem., 270, p. 15870 (1995);ICH-1 (or Nedd-2) (Wang, L. et al., Cell, 78, p. 739 (1994)), MCH-2,(Fernandes-Alnemri, T. et al., Cancer Res., 55, p. 2737 (1995), CPP32(or YAMA or apopain) (Fernandes-Alnemri, T. et al., J. Biol. Chem., 269,p. 30761 (1994); Nicholson, D. W. et al., Nature, 376, p. 37 (1995)),and CMH-1 (or MCH-3) (Lippke, et al., J. Biol. Chem., (1996);Fernandes-Alnemri, T. et al., Cancer Res., (1995)). Each of these ICEhomologs, as well as ICE itself, is capable of inducing apoptosis whenoverexpressed in transfected cell lines. Inhibition of one or more ofthese homologs with the peptidyl ICE inhibitorTyr-Val-Ala-Asp-chloromethylketone results in inhibition of apoptosis inprimary cells or cell lines. Lazebnik et al., Nature, 371, p. 346(1994). The compounds described herein are also capable of inhibitingone or more homologs of ICE. Therefore, these compounds may be used toinhibit apoptosis in tissue types that contain ICE homologs.

Interferon-gamma inducing factor (IGIF) is an approximately 18-kDapolypeptide that stimulates T-cell production of interferon-gamma(IFN-γ). IGIF is produced by activated Kupffer cells and macrophages invivo and is exported out of such cells upon endotoxin stimulation. Thus,a compound that decreases IGIF production would be useful as aninhibitor of such T-cell stimulation which in turn would reduce thelevels of IFN-γ production by those cells.

IFN-γ is a cytokine with immunomodulatory effects on a variety of immunecells. In particular, IFN-γ is involved in macrophage activation and Th1cell selection (F. Belardelli, APMIS, 103, p. 161 (1995)). IFN-γ exertsits effects in part by modulating the expression of genes through theSTAT and IRF pathways (C. Schindler and J. E. Darnell, Ann. Rev.Biochem., 64, p. 621 (1995); T. Taniguchi, J. Cancer Res. Clin. Oncol.,121, p. 516 (1995)).

Mice lacking IFN-γ or its receptor have multiple defects in immune cellfunction and are resistant to endotoxic shock (S. Huang et al., Science,259, p.1742 (1993); D. Dalton et al., Science, 259, p.1739 (1993); B. D.Car et al., J. Exp. Med., 179, p.1437 (1994)). Along with IL-12, IGIFappears to be a potent inducer of IFN-γ production by T cells (H.Okamura et al., Infection and Immunity, 63, p.3966 (1995); H. Okamura etal., Nature, 378, p.88 (1995); S. Ushio et al., J. Immunol., 156, p.4274(1996)).

IFN-γ has been shown to contribute to the pathology associated with avariety of inflammatory, infectious and autoimmune disorders anddiseases. Thus, compounds capable of decreasing IFN-γ production wouldbe useful to ameliorate the effects of IFN-γ related pathologies.

IGIF is synthesized as a precursor protein, called “pro-IGIF”. Recently,ICE and other members of the ICE/CED-3 family have been linked to theconversion of pro-IGIF to IGIF or to IFN-γ production in vivo (see PCTpatent application WO 97/22619, which is incorporated herein byreference).

Accordingly, compositions and methods capable of regulating theconversion of pro-IGIF to IGIF would be useful for decreasing IGIF andIFN-γ production in vivo, and thus for ameliorating the detrimentaleffects of these proteins which contribute to human disorders anddiseases.

ICE inhibitors represent a class of compounds useful for the control ofinflammation or apoptosis or both. Peptide and peptidyl inhibitors ofICE have been described. PCT patent applications WO 91/15577; WO93/05071; WO 93/09135; WO 93/14777 and WO 93/16710; and European patentapplication 0 547 699. Such peptidyl inhibitors of ICE have beenobserved to block the production of mature IL-1β in a mouse model ofinflammation (vide infra) and to suppress growth of leukemia cells invitro (Estrov et al., Blood 84, 380a (1994)). However, due to theirpeptidic nature, such inhibitors are typically characterized byundesirable pharmacologic properties, such as poor cellular penetrationand cellular activity, poor oral absorption, poor stability and rapidmetabolism. Plattner, J. J. and D. W. Norbeck, in Drug DiscoveryTechnologies, C. R. Clark and W. H. Moos, Eds. (Ellis Horwood,Chichester, England, 1990), pp.92-126. This has hampered theirdevelopment into effective drugs.

Non-peptidyl compounds have also been reported to inhibit ICE in vitro.PCT patent application WO 95/26958; U.S. Pat. No. 5,552,400; Dolle etal., J. Med. Chem., 39, pp. 2438-2440 (1996). However, it is not clearwhether these compounds have the appropriate pharmacological profiles tobe therapeutically useful.

Additionally, current methods for the preparation of such compounds arenot advantageous. These methods use tributyltin hydride, a toxic,moisture-sensitive reagent. Thus, these methods are inconvenient tocarry out, pose a health risk and create toxic-waste disposal problems.Furthermore, it is difficult to purify compounds prepared by thesemethods. A preferred method for preparing compounds, such as the ICEinhibitors of this invention, has been described in PCT patentapplication WO 97/22619, which is incorporated herein by reference.

Accordingly, the need exists for compounds that can effectively inhibitthe action of ICE in vivo, for use as agents for preventing and treatingchronic and acute forms of IL-1-mediated diseases, apoptosis-, IGIF-, orIFN-γ-mediated diseases, as well as inflammatory, autoimmune,destructive bone, proliferative, infectious, or degenerative diseases.The need also exists for methods of preparing such compounds.

SUMMARY OF THE INVENTION

The present invention provides novel classes of compounds, andpharmaceutically acceptable derivatives thereof, that are useful asinhibitors of ICE. These compounds can be used alone or in combinationwith other therapeutic or prophylactic agents, such as antibiotics,immunomodulators or other anti-inflammatory agents, for the treatment orprophylaxis of diseases mediated by IL-1, apoptosis, IGIF or IFN-γ.According to a preferred embodiment, the compounds of this invention arecapable of binding to the active site of ICE and inhibiting the activityof that enzyme. Additionally, they have improved cellular potency,improved pharmacokinetics, and/or improved oral bioavailability comparedto peptidyl ICE inhibitors.

It is a principal object of this invention to provide novel classes ofcompounds which are inhibitors of ICE represented by formula:

wherein the various substituents are described herein.

It is a further objective of this invention to provide novel processesof preparing the compounds of this invention and related compounds.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention described herein may be more fullyunderstood, the following detailed description is set forth.

The following abbreviations and definitions are used throughout theapplication.

Abbreviations Ac₂O acetic anhydride n-Bu normal-butyl D MFdimethylformamide DIEA N,N-diisopropylethylamine EDC1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Et₂O diethylether EtOAc ethyl acetate Fmoc 9-fluorenylmethyoxycarbonyl HBTUO-benzotriazol-1-yl-N,N,N,N′-tetramethyluronium hexafluorophosphate HOBT1-hydroxybenzotriazole hydrate MeOH methanol TFA trifluoroacetic acid

The terms “HBV”, “HCV” and “HGV” refer to hepatitis-B virus, hepatitis-Cvirus and hepatitis-G virus, respectively.

The term “K_(i)” refers to a numerical measure of the effectiveness of acompound in inhibiting the activity of a target enzyme such as ICE.Lower values of K_(i) reflect higher effectiveness. The K_(i) value is aderived by fitting experimentally determined rate data to standardenzyme kinetic equations (see I. H. Segel, Enzyme Kinetics,Wiley-Interscience, 1975).

The term “interferon gamma inducing factor” or “IGIF” refers to a factorwhich is capable of stimulating the endogenous production of IFN-γ.

The term “ICE inhibitor” refers to a compound which is capable ofinhibiting one or more enzymes selected from the group consisting of ICEand ICE homologs. ICE inhibition may be determined using the methodsdescribed and incorporated by reference herein. The skilled practitionerrealizes that an in vivo ICE inhibitor is not necessarily an in vitroICE inhibitor. For example, a prodrug form of a compound typicallydemonstrates little or no activity in in vitro assays. Such prodrugforms may be altered by metabolic or other biochemical processes in thepatient to provide an in vivo ICE inhibitor.

The term “cytokine” refers to a molecule which mediates interactionsbetween cells.

The term “condition” refers to any disease, disorder or effect thatproduces deleterious biological consequences in a subject.

The term “subject” refers to an animal, or to one or more cells derivedfrom an animal. Preferably, the animal is a mammal, most preferably ahuman. Cells may be in any form, including but not limited to cellsretained in tissue, cell clusters, immortalized cells, transfected ortransformed cells, and cells derived from an animal that have beenphysically or phenotypically altered.

The term “patient” as used in this application refers to any mammal,preferably humans

The term “alkyl” refers to a straight-chained or branched, saturatedaliphatic hydrocarbon containing 1 to 6 carbon atoms.

The term “alkenyl” refers to a straight-chained or branched unsaturatedhydrocarbon containing 2 to 6 carbons.

The term “cycloalkyl” refers to a mono- or polycyclic, non-aromatic,hydrocarbon ring system which may optionally contain unsaturated bondsin the ring system. Examples include cyclohexyl, adamantyl andnorbornyl.

The term “aryl” refers to a mono- or polycyclic ring system whichcontains 6, 10, 12 or 14 carbons in which at least one ring of the ringsystem is aromatic. The aryl groups of this invention are optionallysingly or multiply substituted with R¹⁷. Examples of aryl ring systemsinclude, phenyl, naphthyl, and tetrahydronaphthyl.

The term “heteroaryl” refers to a mono- or polycyclic ring system whichcontains 1 to 15 carbon atoms and 1 to 4 heteroatoms, and in which atleast one ring of the ring system is aromatic. Heteroatoms are sulfur,nitrogen or oxygen. The heteroaryl groups of this invention areoptionally singly or multiply substituted with R¹⁷.

The term “heterocyclic” refers to a mono- or polycyclic ring systemwhich contains 1 to 15 carbon atoms and 1 to 4 heteroatoms, in which themono- or polycyclic ring system may optionally contain unsaturated bondsbut is not aromatic. Heteroatoms are independently sulfur, nitrogen, oroxygen.

The term “alkylaryl” refers to an alkyl group, wherein a hydrogen atomof the alkyl group is replaced by an aryl radical.

The term “alkylheteroaryl” refers to an alkyl group, wherein a hydrogenatom of the alkyl group is replaced by a heteroaryl radical.

The term “substitute” refers to the replacement of a hydrogen atom in acompound with a substituent group.

The term “straight chain” refers to a contiguous unbranching string ofcovalently bound atoms. The straight chain may be substituted, but thesesubstituents are not a part of the straight chain.

The term “patient” as used in this application refers to any mammal,preferably humans.

In chemical formulas, parenthesis are used herein to denote connectivityin molecules or groups. In particular, parentheses are used toindicate: 1) that more than one atom or group is bonded to a particularatom; or 2) a branching point (i.e., the atom immediately before theopen parenthesis is bonded both to the atom or group in the parenthesesand the atom or group immediately after the closed parenthesis). Anexample of the first use is “—N(alkyl)₂”, indicating two alkyl groupsbond to an N atom. An example of the second use is “—C(O)NH₂”,indicating a carbonyl group and an amino (“NH₂”) group both bonded tothe indicated carbon atom. A “—C(O)NH₂” group may be represented inother ways, including the following structure:

Other definitions are set forth in the specification where necessary.

Compounds of this Invention

The compounds of one embodiment (A) of this invention are those offormula (I):

wherein:

Y is

provided that if R⁵ is —OH, then Y may also be:

C is an aryl or a heteroaryl ring, wherein any hydrogen bound to anyring atom is optionally replaced by —R⁴;

R¹ is -aryl, -heteroaryl, -alkylaryl, or -alkylheteroaryl;

R² is a bond, —C(O)—, —C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—,—N(H)S(O)₂—, —N(H)C(O)C(O)—, —CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—,—N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—, —N(R¹⁹)C(O)C(O)—, or —N(R¹⁹)CH₂C(O)—,provided that if R² is not a bond, then R² is connected to the NHattached to the 7-membered ring through carbonyl or sulfonyl;

R³ is -aryl, -heteroaryl, -cycloalkyl, -alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H)alkyl, —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O)₂alkyl, or —C(O)alkyl;

R⁵ is —OH, —OR⁸, or —N(H)OH;

R⁶ is —H, —CH₂OR⁹, —CH₂SR¹⁰, —CH₂N(H)R⁹, —CH₂N(R⁹)R¹², —C(H)N₂, —CH₂F,—CH₂Cl, —C(O)N(R¹¹)R¹², —R¹³, or —R¹⁴;

R⁸ is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

R⁹ is —H, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl,—C(O)alkylheteroaryl, -alkylaryl, -alkylheteroaryl, -aryl, -heteroaryl,or —P(O)R¹⁵R¹⁶;

R¹⁰ is -alkylaryl, -aryl, -heteroaryl, or -alkylheteroaryl;

each R¹¹ and R¹² is independently —H, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, or -alkylheteroaryl;

R¹³ is -alkylaryl, -alkenylaryl, -alkynylaryl, or -alkylheteroaryl;

R¹⁴ is

wherein any hydrogen bound to (i) is optionally replaced with R¹⁷ andany hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ or R²⁰;

each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, -alkylheteroaryl, —Oalkyl, —Oaryl,—Oheteroaryl, —Oalkylaryl, or —Oalkylheteroaryl;

R¹⁷ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, —SO₂NH₂, —C(O)H, -alkyl, -cycloalkyl,-perfluoroalkyl, —O-alkyl, —N(H)alkyl, —N(alkyl)₂, —CO₂alkyl,—C(O)N(H)alkyl, —C(O)N(alkyl)₂, —N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl,—N(H)C(O)N(alkyl)₂, —S(O₂)N(H)alkyl, —S(O₂)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, or —C(O)alkyl;

R¹⁸ is -aryl, -heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl,—O-heteroaryl, —O-alkylaryl, —O-alkylheteroaryl, —N(H)aryl, —N(aryl)₂,—N(H)heteroaryl, —N(heteroaryl)₂, —N(H)alkylaryl, —N(alkylaryl)₂,—N(H)alkylheteroaryl, —N(alkylheteroaryl)₂, —S-aryl, —S-heteroaryl,—S-alkylaryl, —S-alkylheteroaryl, —C(O)aryl, —C(O)heteroaryl,—C(O)alkylaryl, —C(O)alkyheteroaryl, —CO₂aryl, —CO₂heteroaryl,—CO₂alkylaryl, —CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂,—C(O)N(H)heteroaryl, —C(O)N(heteroaryl)₂, —C(O)N(H)alkylaryl,—C(O)N(alkylaryl)₂, —C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂,—S(O)₂aryl, —S(O)₂heteroaryl, —S(O)₂alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂N(H)aryl, —S(O)₂N(H)heteroaryl, —S(O)₂N(H)alkylaryl,—S(O)₂N(H)alkylheteroaryl, —S(O)₂N(aryl)₂, —S(O)₂N(heteroaryl)₂,—S(O)₂N(alkylaryl)₂, —S(O)₂N(alkylheteroaryl)₂, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroaryl, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂,—N(H)C(O)N(alkylheteroaryl)₂;

R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

R²⁰ is -alkyl—R¹⁸;

m is 0 or 1; and

X is O or S.

The compounds of another embodiment (B) of this invention are those offormula (II):

wherein Y is:

R⁷ is —C(O)alkyl, —C(O)cycloalkyl, —C(O)alkyenyl, —C(O)alkylaryl,—C(O)alkylheteroaryl, —C(O)heterocycle, or —C(O)alkylheterocycle; andthe other substituents are as described above.

Preferred compounds of embodiments (A) and (B) are those wherein:

C is benzo, pyrido, thieno, pyrrolo, furo, imidazo, thiazolo, oxazolo,pyrazolo, isothiazolo, isoxazolo, or triazolo, wherein any hydrogenbound to any ring atom is optionally replaced by R⁴.

More preferred compounds of embodiments (A) and (B) are those wherein:

Y is

C is benzo, wherein any hydrogen bound to any ring atom is optionallyreplaced by R⁴;

R¹ is -phenyl, -naphthyl, or -isoquinolinyl, wherein R¹⁷ is —OH, —NH₂,—Cl, —F, —Oalkyl, or —N(alkyl)₂;

R² is —C(O)—, —S(O₂)—, —C(O)C(O)—, or —CH₂C(O)—;

R³ is -methyl, -ethyl, -n-propyl, -isopropyl, -phenyl, -2-pyridinyl,-3-pyridinyl, -4-pyridinyl, or -thiazolyl;

R⁴ is -fluoro or -chloro;

R⁵ is —OH;

R⁶ is:

—H; or

—R¹⁴, wherein X═O, provided that when —R¹⁴ is (i), R¹⁷ is —Oalkyl, —F or—Cl and provided that when —R¹⁴ is (ii), R¹⁸ is -aryl, wherein aryl isphenyl;

R⁷ is —C(O)alkyl;

R⁸ is -methyl, -ethyl, -n-propyl, -isopropyl, -cyclopentyl, -phenethylor -benzyl;

X is O; or

m is 0.

Preferred compounds of embodiment (A) of this invention include, but arenot limited to:

We now prefer compounds of embodiments (C) and (D). The compounds ofembodiment (C) of this invention are those of formula (III):

wherein:

Y is

provided that if R⁵ is —OH, then Y may also be:

C is an aryl or a heteroaryl ring, wherein any hydrogen bound to anyring atom is optionally replaced by —R⁴;

R¹ is -aryl, -heteroaryl, -alkylaryl, or -alkylheteroaryl;

R² is a bond, —C(O)—, —C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—,—N(H)S(O)₂—, —N(H)C(O)C(O)—, —CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—,—N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—, —N(R¹⁹)C(O)C(O)—, —N(R¹⁹)CH₂C(O)—, or—C(O)C(═NOR¹¹)—, provided that when R² is not a bond, R² is bonded tothe 7-membered ring NH group through carbonyl or sulfonyl;

R³ is -aryl, -heteroaryl, -cycloalkyl, -alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H)(alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or CH₂N(alkyl)₂;

R⁵ is —OH, —OR⁸, or —N(H)OH;

R⁶ is —H, —CH₂OR⁹, —CH₂SR¹⁰, —CH₂NHR⁹, —CH₂N(R⁹)R¹², —CHN₂, —CH₂F,—CH₂Cl, —C(O)N(R¹¹)R¹², —R¹³, or —R¹⁴;

R⁸ is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

R⁹ is —H, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl,—C(O)alkylheteroaryl, -alkylaryl, -alkylheteroaryl, -aryl, -heteroaryl,or —P(O)R¹⁵R¹⁶;

R¹⁰ is -alkylaryl, -aryl, -heteroaryl, or -alkylheteroaryl;

each R¹¹ and R¹² is independently —H, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, or -alkylheteroaryl;

R¹³ is -alkylaryl, -alkenylaryl, -alkynylaryl, or -alkylheteroaryl;

R¹⁴ is

wherein any hydrogen bound to (i) is optionally replaced with R¹⁷ andany hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ or R²⁰;

each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, -alkylheteroaryl, —Oalkyl, —Oaryl,—Oheteroaryl, —Oalkylaryl, or —Oalkylheteroaryl;

R¹⁷ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, —S(O₂)NH₂, —C(O)H, -alkyl, -cycloalkyl,-perfluoroalkyl, —O-alkyl, —N(H)alkyl, —N(alkyl)₂, —CO₂alkyl,—C(O)N(H)alkyl, —C(O)N(alkyl)₂, —N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl,—N(H)C(O)N(alkyl)₂, —SO₂N(H)alkyl, —S(O₂)N(alkyl)₂, —S—alkyl,—S(O₂)alkyl, or —C(O)alkyl;

R¹⁸ is -aryl, -heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl,—O-heteroaryl, —O-alkylaryl, —O-alkylheteroaryl, —N(H)aryl, —N(aryl)₂,—N(H)heteroaryl, —N(heteroaryl)₂, —N(H)alkylaryl, —N(alkylaryl)₂,—N(H)alkylheteroaryl, —N(alkylheteroaryl)₂, —S-aryl, —S-heteroaryl,—S-alkylaryl, —S-alkylheteroaryl, —C(O)aryl, —C(O)heteroaryl,—C(O)alkylaryl, —C(O)alkyheteroaryl, —CO₂aryl; —CO₂heteroaryl,—CO₂alkylaryl, —CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂,—C(O)N(H)heteroaryl, —C(O)N(heteroaryl)₂, —C(O)N(H)alkylaryl,—C(O)N(alkylaryl)₂, —C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂,—S(O)₂-aryl, —S(O)₂-heteroaryl, —S(O)₂-alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂NH-aryl, —S(O)₂NH—heteroaryl, —S(O)₂N(H)alkylaryl,—S(O)₂N(H)alkylheteroaryl, —S(O)₂N(aryl)₂, —S(O)₂N(H)heteroaryl)₂,—SO₂N(alkylaryl)₂, —SO₂N(alkylheteroaryl)₂, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroaryl, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(H)heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂,—N(H)C(O)N(alkylheteroaryl)₂;

R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or -alkylheterocycle;

R²⁰ is -alkyl-R¹⁸;

m is 0 or 1; and

X is O or S.

The compound of embodiment (D) of this invention are those of formula(IV):

wherein Y is:

R⁷ is —C(O)alkyl, —C(O)cycloalkyl, —C(O)alkyenyl, —C(O)alkylaryl,—C(O)alkylheteroaryl, —C(O)heterocycle, or —C(O)alkylheterocycle; andthe other substituents are as defined above.

In the above embodiments, R⁸ and R¹⁹ is also independently selected fromheterocyclyl or alkylcycloalkyl.

In embodiments (B) and (D) Y is also selected from:

Preferred compounds of embodiments (C) and (D) are those wherein:

C is benzo, pyrido, thieno, pyrrolo, furo, imidazo, thiazolo, oxazolo,pyrazolo, isothiazolo, isoxazolo, or triazolo, wherein any hydrogenbound to any ring atom is optionally replaced by R⁴.

More preferred compounds of embodiment (C) and (D) are those wherein:

Y is

C is benzo, wherein any hydrogen bound to any ring atom is optionallyreplaced by R⁴;

R¹ is -phenyl, -naphthyl, or -isoquinolinyl, wherein R¹⁷ is —OH, —NH₂,—Cl, —F, —Oalkyl, or —N(alkyl)₂;

R² is —C(O)—, —S(O)₂—, —C(O)C(O)—, or —CH₂C(O)—

R³ is -methyl, -ethyl, -n-propyl, -isopropyl, -phenyl, -2-pyridinyl,-3-pyridinyl, -4-pyridinyl, or -thiazolyl;

R⁴ is -fluoro or -chloro;

R⁵ is —OH;

R⁶ is:

—H; or

—R¹⁴, wherein X is O, provided that when —R¹⁴ is (i), R¹⁷ is —Oalkyl, —For —Cl and provided that when —R¹⁴ is (ii), R¹⁸ is -aryl, wherein arylis phenyl;

R⁷ is —C(O)alkyl;

R⁸ is -methyl, -ethyl, -n-propyl, -isopropyl, -cyclopentyl, -phenethylor -benzyl;

X is O; or

m is 0.

Other more preferred compounds of embodiments (B) and (D) are thosewherein Y is

and the other substituents are as defined above.

Preferred compounds of this invention include, but are not limited to:

Another preferred compound of this invention includes, but is notlimited to:

The ICE inhibitors of this invention may contain one or more“asymmetric” carbon atoms and thus may occur as racemates and racemicmixtures, single enantiomers, diastereomeric mixtures and individualdiastereomers. All such isomeric forms of these compounds are expresslyincluded in the present invention. Each stereogenic carbon may be of theR or S configuration. Although specific compounds and scaffoldsexemplified in this application may be depicted in a particularstereochemical configuration, compounds and scaffolds having either theopposite stereochemistry at any given chiral center or mixtures thereofare also envisioned.

The ICE inhibitors may optionally be substituted at carbon, nitrogen orother atoms by various substituents. When multiply substituted, eachsubstituent may be picked independently of any other substituent as longas the combination of substituents results in the formation of a stablecompound.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and administration to a mammalby methods known in the art. Typically, such compounds are stable at atemperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

Substituents may be represented in various forms. These various formsare known to the skilled practitioner and may be used interchangeably.For example, a methyl substituent on a phenyl ring may be represented inany of the following forms:

Various forms of substituents such as methyl are used hereininterchangeably.

The compounds of this invention have a molecular weight of less than orequal to about 700 Daltons, and more preferably between about 400 and600 Daltons. These preferred compounds may be readily absorbed by thebloodstream of patients upon oral administration. This oral availabilitymakes such compounds excellent agents for orally-administered treatmentand prevention regimens against IL-1-, apoptosis-, IGIF-, orIFN-γ-mediated diseases.

It should be understood that the compounds of this invention may existin various equilibrium forms, depending on conditions including choiceof solvent, pH, and others known to the practitioner skilled in the art.All such forms of these compounds are expressly included in the presentinvention. In particular, many of the compounds of this invention,especially those which contain aldehyde or ketone groups in R₃ andcarboxylic acid groups in T, may take hemi-ketal (or hemi-acetal) orhydrated forms. For example, compounds of embodiment (A) take ahemiacetal or hemiketal form when Y is:

Depending on the choice of solvent and other conditions known to thepractitioner skilled in the art, compounds of this invention may alsotake hydrated, acyloxy ketal, acyloxy acetal, ketal, acetal or enolforms. For example, in embodiment (B) compounds of this invention takehydrated forms when Y is:

acyloxy ketal or acyloxy acetal forms when Y is:

ketal or acetal forms when Y is:

and enol forms when Y is:

In addition, it should be understood that the equilibrium forms of thecompounds of this invention may include tautomeric forms. All such formsof these compounds are expressly included in the present invention.

It should be understood that the compounds of this invention may bemodified by appropriate functionalities to enhance selective biologicalproperties. Such modifications are known in the art and include thosewhich increase biological penetration into a given biological system(e.g., blood, lymphatic system, central nervous system), increase oralavailability, increase solubility to allow administration by injection,alter metabolism and alter rate of excretion. In addition, the compoundsmay be altered to pro-drug form such that the desired compound iscreated in the body of the patient as the result of the action ofmetabolic or other biochemical processes on the pro-drug. Such pro-drugforms typically demonstrate little or no activity in in vitro assays.Some examples of pro-drug forms include ketal, acetal, oxime, imine andhydrazone forms of compounds which contain ketone or aldehyde groups,especially where they occur in the R³ group of the compounds of thisinvention. Other examples of pro-drug forms include the hemi-ketal,hemi-acetal, acyloxy ketal, acyloxy acetal, ketal, acetal and enol formsthat are described herein.

Compositions and Methods

The compounds of this invention are excellent ligands for ICE.Accordingly, these compounds are capable of targeting and inhibitingevents in IL-1-, apoptosis-, IGIF- and IFN-γ-mediated diseases, and,thus, the ultimate activity of that protein in inflammatory diseases,autoimmune diseases, destructive bone, proliferative disorders,infectious diseases, and degenerative diseases. For example, thecompounds of this invention inhibit the conversion of precursor IL-1β tomature IL-1β by inhibiting ICE. Because ICE is essential for theproduction of mature IL-1, inhibition of that enzyme effectively blocksinitiation of IL-1-mediated physiological effects and symptoms, such asinflammation, by inhibiting the production of mature IL-1. Thus, byinhibiting IL-1β precursor activity, the compounds of this inventioneffectively function as IL-1 inhibitors.

Compounds of this invention also inhibit conversion of pro-IGIF intoactive, mature IGIF by inhibiting ICE. Because ICE is essential for theproduction of mature IGIF, inhibition of ICE effectively blocksinitiation of IGIF-mediated physiological effects and symptoms, byinhibiting production of mature IGIF. IGIF is in turn essential for theproduction of IFN-γ. ICE therefore effectively blocks initiation ofIFN-γ-mediated physiological effects and symptoms, by inhibitingproduction of mature IGIF and thus production of IFN-γ.

The pharmaceutical compositions and methods of this invention,therefore, will be useful for controlling ICE activity in vivo. Thecompositions and methods of this invention will thus be useful forcontrolling IL-1, IGIF or IFN-γ levels in vivo and for treating orreducing the advancement, severity or effects of IL-1-, apoptosis-,IGIF-, or IFN-γ-mediated conditions, including diseases, disorders oreffects.

Accordingly, one embodiment of this invention provides a method fordecreasing IGIF production in a subject comprising the step ofadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of an ICE inhibitor and apharmaceutically acceptable carrier.

Another embodiment of this invention provides a method for decreasingIFN-γ production in a subject comprising the step of administering tothe subject a pharmaceutical composition comprising a therapeuticallyeffective amount of an ICE inhibitor and a pharmaceutically acceptablecarrier.

In another embodiment, the methods of this invention comprise the stepof administering to a subject a pharmaceutical composition comprising aninhibitor of an ICE-related protease that is capable of cleavingpro-IGIF to active IGIF, and a pharmaceutically acceptable carrier. Onesuch ICE-related protease is TX, as described above. This invention thusprovides methods and pharmaceutical compositions for controlling IGIFand IFN-γ levels by administering a TX inhibitor.

Other ICE-related proteases capable of processing pro-IGIF into anactive IGIF form may also be found. Thus it is envisioned thatinhibitors of those enzymes may be identified by those of skill in theart and will also fall within the scope of this invention.

Pharmaceutical compositions of this invention comprise an ICE inhibitoror a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier, adjuvant or vehicle. Such compositions mayoptionally comprise an additional therapeutic agent. Such agentsinclude, but are not limited to, an anti-inflammatory agent, a matrixmetalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator, a prostaglandinor an anti-vascular hyperproliferation compound.

If the pharmaceutical composition comprises only the ICE inhibitor asthe active component, such methods may additionally comprise the step ofadministering to the subject an additional agent. Such agents include,but are not limited to, an anti-inflammatory agent, a matrixmetalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator, a prostaglandinor an anti-vascular hyperproliferation compound.

The term “pharmaceutically effective amount” refers to an amounteffective in treating or ameliorating an IL-1-, apoptosis-, IGIF- orIFN-γ-mediated disease in a patient. The term “prophylacticallyeffective amount” refers to an amount effective in preventing orsubstantially lessening IL-1-, apoptosis-, IGIF- or IFN-γ-mediateddiseases in a patient.

The term “pharmaceutically acceptable carrier or adjuvant” refers to anon-toxic carrier or adjuvant that may be administered to a patient,together with a compound of this invention, and which does not destroythe pharmacological activity thereof.

The term “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ester, or salt of such ester, of acompound of this invention or any other compound which, uponadministration to a recipient, is capable of providing (directly orindirectly) a compound of this invention or an anti-ICE activemetabolite or residue thereof.

Pharmaceutically acceptable salts of the compounds of this inventioninclude, for example, those derived from pharmaceutically acceptableinorganic and organic acids and bases. Examples of suitable acidsinclude hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic,benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids.Other acids, such as oxalic, while not in themselves pharmaceuticallyacceptable, may be employed in the preparation of salts useful asintermediates in obtaining the compounds of the invention and theirpharmaceutically acceptable acid addition salts. Salts derived fromappropriate bases include alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N—(C₁₋₄ alkyl)₄ ⁺ salts.

This invention also envisions the “quaternization” of any basicnitrogen-containing groups of the compounds disclosed herein. The basicnitrogen can be quaternized with any agents known to those of ordinaryskill in the art including, for example, lower alkyl halides, such asmethyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkylsulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides including benzyl and phenethylbromides. Water or oil-soluble or dispersible products may be obtainedby such quaternization.

The compounds of this invention may be employed in a conventional mannerfor controlling IGIF and IFN-γ levels in vivo and for treating diseasesor reducing the advancement or severity of effects which are mediated byIL-1, apoptosis, IGIF or IFN-γ. Such methods of treatment, their dosagelevels and requirements may be selected by those of ordinary skill inthe art from available methods and techniques.

For example, a compound of this invention may be combined with apharmaceutically acceptable adjuvant for administration to a patientsuffering from an IL-1-, apoptosis-, IGIF- or IFN-γ-mediated disease ina pharmaceutically acceptable manner and in an amount effective tolessen the severity of that disease.

Alternatively, the compounds of this invention may be used incompositions and methods for treating or protecting individuals againstIL-1, apoptosis-, IGIF, or IFN-γ mediated diseases over extended periodsof time. The compounds may be employed in such compositions either aloneor together with other compounds of this invention in a mannerconsistent with the conventional utilization of ICE inhibitors inpharmaceutical compositions. For example, a compound of this inventionmay be combined with pharmaceutically acceptable adjuvantsconventionally employed in vaccines and administered in prophylacticallyeffective amounts to protect individuals over an extended period of timeagainst IL-1-, apoptosis-, IGIF, or IFN-γ mediated diseases.

The compounds of this invention may also be co-administered with otherICE inhibitors to increase the effect of therapy or prophylaxis againstvarious IL-1-, apoptosis-, IGIF- or IFN-γ mediated diseases.

In addition, the compounds of this invention may be used in combinationeither conventional anti-inflammatory agents or with matrixmetalloprotease inhibitors, lipoxygenase inhibitors and antagonists ofcytokines other than IL-1β.

The compounds of this invention can also be administered in combinationwith immunomodulators (e.g., bropirimine, anti-human alpha-interferonantibody, IL-2, GM-CSF, methionine enkephalin, interferon-alpha,diethyldithiocarbamate, tumor necrosis factor, naltrexone and EPO), withprostaglandins, or with antiviral agents (e.g., 3TC, polysulfatedpolysaccharides, ganiclovir, ribavirin, acyclovir, alpha interferon,trimethotrexate and fancyclovir) or prodrugs of these or relatedcompounds to prevent or combat IL-1-mediated disease symptoms such asinflammation.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to the patient. Alternatively, pharmaceutical orprophylactic compositions according to this invention comprise acombination of an ICE inhibitor of this invention and anothertherapeutic or prophylactic agent.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat and self-emulsifying drug delivery systems (SEDDS) such asα-tocopherol, polyethyleneglycol 1000 succinate, or other similarpolymeric delivery matrices.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. We prefer oraladministration. The pharmaceutical compositions of this invention maycontain any conventional non-toxic pharmaceutically-acceptable carriers,adjuvants or vehicles. In some cases, the pH of the formulation may beadjusted with pharmaceutically acceptable acids, bases or buffers toenhance the stability of the formulated compound or its delivery form.The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as those described in Pharmacopeia Helvetica, Ph.Helv, or a similar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-administered transdermalpatches are also included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably between 0.5 and about 75 mg/kg body weight per day andmost preferably between about 1 and 50 mg/kg body weight per day of theactive ingredient compound are useful in a monotherapy for theprevention and treatment of IL-1-, apoptosis-, IGIF- and IFN-γ mediateddiseases, including inflammatory diseases, autoimmune diseases,destructive bone disorders, proliferative disorders, infectiousdiseases, degenerative diseases, necrotic diseases, osteoarthritis,acute pancreatitis, chronic pancreatitis, asthma, adult respiratorydistress syndrome, glomerulonephritis, rheumatoid arthritis, systemiclupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease,autoimmune gastritis, insulin-dependent diabetes mellitus (Type I),autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia,chronic active hepatitis, myasthenia gravis, inflammatory bowel disease,Crohn's disease, psoriasis, graft vs. host disease, osteoporosis,multiple myeloma-related bone disorder, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma,multiple myeloma, sepsis, septic shock, Shigellosis, Alzheimer'sdisease, Parkinson's disease, cerebral ischemia, myocardial ischemia,spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis,HIV-related encephalitis, aging, alopecia, neurological damage due tostroke, ulcerative collitis, infectious hepatitis, juvenile diabetes,lichenplanus, acute dermatomyositis, eczema, primary cirrhosis, uveitis,Behcet's disease, atopic skin disease, pure red cell aplasia, aplasticanemia, amyotrophic lateral sclerosis, nephrotic syndrome and systemicdiseases or diseases with effects localized in the liver or other organshaving an inflammatory or apoptotic component caused by excess dietaryalcohol intake or viruses, such as HBV, HCV, HGV, yellow fever virus,dengue fever virus, and Japanese encephalitis virus.

Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to 5 times per day or alternatively, as acontinuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Preferably, such preparations containfrom about 20% to about 80% active compound.

When the compositions of this invention comprise a combination of an ICEinhibitor and one or more additional therapeutic or prophylactic agents,both the ICE inhibitor and the additional agent should be present atdosage levels of between about 10% to 80% of the dosage normallyadministered in a monotherapy regime.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence or disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of the disease,and the patient's disposition to the disease and the judgment of thetreating physician.

The IL-1 mediated diseases which may be treated or prevented by thecompounds of this invention include, but are not limited to,inflammatory diseases, autoimmune diseases, proliferative disorders,infectious diseases, and degenerative diseases. The apoptosis-meditateddiseases which may be treated or prevented by the compounds of thisinvention include degenerative diseases.

IL-1 mediated inflammatory diseases which may be treated or preventedinclude, but are not limited to osteoarthritis, acute pancreatitis,chronic pancreatitis, asthma, and adult respiratory distress syndrome.Preferrably the inflammatory disease is osteoarthritis or acutepancreatitis.

IL-1 mediated autoimmune diseases which may be treated or preventedinclude, but are not limited to, glomeralonephritis, rheumatoidarthritis, systemic lupus erythematosus, scleroderma, chronicthyroiditis, Graves' disease, autoimmune gastritis, insulin-dependentdiabetes mellitus (Type I), autoimmune hemolytic anemia, autoimmuneneutropenia, thrombocytopenia, chronic active hepatitis, myastheniagravis, multiple sclerosis, inflammatory bowel disease, Crohn's disease,psoriasis, and graft vs. host disease. Preferrably the autoimmunedisease is rheumatoid arthritis, inflammatory bowel disease, Crohn'sdisease, or psoriasis,

IL-1 mediated destructive bone disorders which may be treated orprevented include, but are not limited to, osteoporosis and multiplemyeloma-related bone disorder.

IL-1 mediated proliferative diseases which may be treated or preventedinclude, but are not limited to, acute myelogenous leukemia, chronicmyelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, andmultiple myeloma.

IL-1 mediated infectious diseases which may be treated or preventedinclude, but are not limited to, sepsis, septic shock, and Shigellosis.

The IL-1 mediated degenerative or necrotic diseases which may be treatedor prevented by the compounds of this invention include, but are notlimited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia,and myocardial ischemia. Preferably, the degenerative disease isAlzheimer's disease.

The apoptosis-mediated degenerative diseases which may be treated orprevented by the compounds of this invention include, but are notlimited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia,myocardial ischemia, spinal muscular atrophy, multiple sclerosis,AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia,and neurological damage due to stroke.

Other diseases having an inflammatory or apoptotic component may betreated or prevented by the compounds of this invention. Such diseasesmay be systemic diseases or diseases with effects localized in the liveror other organs and may be caused by, for example, excess dietaryalcohol intake or viruses, such as HBV, HCV, HGV, yellow fever virus,dengue fever virus, and Japanese encephalitis virus.

The IGIF- or IFN-γ-mediated diseases which may be treated or preventedby the compounds of this invention include, but are not limited to,inflammatory, infectious, autoimmune, proliferative, neurodegenerativeand necrotic conditions.

IGIF- or IFN-γ-mediated inflammatory diseases which may be treated orprevented include, but are not limited to osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, rheumatoid arthritis,inflammatory bowel disease, Crohn's disease, ulcerative collitis,cerebral ischemia, myocardial ischemia and adult respiratory distresssyndrome. Preferrably, the inflammatory disease is rheumatoid arthritis,ulcerative collitis, Crohn's disease, hepatitis or adult respiratorydistress syndrome.

IGIF- or IFN-γ-mediated infectious diseases which may be treated orprevented include, but are not limited to infectious hepatitis, sepsis,septic shock and Shigellosis.

IGIF- or IFN-γ-mediated autoimmune diseases which may be treated orprevented include, but are not limited to glomerulonephritis, systemiclupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease,autoimmune gastritis, insulin-dependent diabetes mellitus (Type I),juvenile diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, myasthenia gravis, multiple sclerosis, psoriasis,lichenplanus, graft vs. host disease, acute dermatomyositis, eczema,primary cirrhosis, hepatitis, uveitis, Behcet's disease, atopic skindisease, pure red cell aplasia, aplastic anemia, amyotrophic lateralsclerosis and nephrotic syndrome. Preferrably, the autoimmune disease isglomerulonephritis, insulin-dependent diabetes mellitus (Type I),juvenile diabetes, psoriasis, graft vs. host disease or hepatitis.

Although this invention focuses on the use of the compounds disclosedherein for preventing and treating IL-1, apoptosis-, IGIF,IFN-γ-mediated diseases, the compounds of this invention can also beused as inhibitory agents for other cysteine proteases.

The compounds of this invention are also useful as commercial reagentswhich effectively bind to ICE or other cysteine proteases. As commercialreagents, the compounds of this invention, and their derivatives, may beused to block proteolysis of a target peptide in biochemical or cellularassays for ICE and ICE homologs or may be derivatized to bind to astable resin as a tethered substrate for affinity chromatographyapplications. These and other uses which characterize commercialcysteine protease inhibitors will be evident to those of ordinary skillin the art.

Process of Preparing N-Acylamino Compounds

The ICE inhibitors of this invention may be synthesized usingconventional techniques. Advantageously, these compounds areconveniently synthesized from readily available starting materials.

The compounds of this invention are among the most readily synthesizedICE inhibitors known. Many of the previously described ICE inhibitorscontain four or more chiral centers and numerous peptide linkages. Therelative ease with which the compounds of this invention can besynthesized represents an advantage in the large scale production ofthese compounds.

For example, compounds of this invention may be prepared using theprocesses described herein. As can be appreciated by the skilledpractitioner, these processes are not the only means by which thecompounds described and claimed in this application may be synthesized.Further methods will be evident to those of ordinary skill in the art.Additionally, the various synthetic steps described herein may beperformed in an alternate sequence or order to give the desiredcompounds.

A preferred method for preparing the N-acylamino compounds of thisinvention comprise the steps of:

a) mixing a carboxylic acid with an N-alloc-protected amine in thepresence of an inert solvent, triphenylphoshine, a nucleophilicscavenger, and tetrakis-triphenyl phosphine palladium(0) at ambienttemperature under an inert atmosphere; and

b) adding to the step a) mixture, HOBT and EDC; and optionallycomprising the further step of:

c) hydrolyzing the step b) mixture in the presence of a solutioncomprising an acid and H₂O, wherein the step b) mixture is optionallyconcentrated, prior to hydrolyzing.

Preferably, the inert solvent is CH₂Cl₂, DMF, or a mixture of CH₂Cl₂ andDMF.

Preferably, the nucleophilic scavenger is dimedone, morpholine,trimethylsilyl dimethylamine, or dimethyl barbituric acid. Morepreferably, the nucleophilic scavenger is trimethylsilyl dimethylamineor dimethyl barbituric acid.

Preferably, the solution comprises trifluoroacetic acid in about 1-90%by weight. More preferably, the solution comprises trifluoroacetic acidin about 20-50% by weight.

Alternatively, the solution comprises hydrochloric acid in about 0.1-30%by weight. More preferably, the solution comprises hydrochloric acid inabout 5-15% by weight.

More preferably, in the above process, the inert solvent is CH₂Cl₂, DMF,or a mixture of CH₂Cl₂ and DMF and the nucleophilic scavenger isdimedone, morpholine, trimethylsilyl dimethylamine, or dimethylbarbituric acid.

Most preferably, in the above process the inert solvent is CH₂Cl₂, DMF,or a mixture of CH₂Cl₂ and DMF and the nucleophilic scavenger istrimethylsilyl dimethylamine or dimethyl barbituric acid.

In an example of a preferred process, the N-acylamino compound isrepresented by formula (V):

wherein:

R²¹ is:

R²² is:

m is 1; and

R²³ is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or alkylheterocycle and the other substituents are asdescribed above.

Preferably, the carboxylic acid is R²²—OH and the N-alloc protectedamine is:

(IV):

wherein R²³ and m are as defined above.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLE 1

(3S)-3-[3(R,S)-1,3-Dihydro-2-oxo-5-phenyl((benzyloxycarbonyl)amino)-2H-1,4-benzodiazepin-1-acetylamino]-4-oxobutyricacid

Step 1. A 0° C. THF solution (30 ml) of 1 (2.4 g 6.22 mmol; prepared asdescribed in Sherrill and Sugg, J. Ore. Chem., 60, pp. 730-4 (1995)) wastreated with NaH (240 mg, 6.00 mmol of a 60% oil dispersion). After thereaction was stirred for 1 hr at 0° C., methyl bromoacetate (0.6 ml,6.32 mmol) was added to the reaction and the allowed to warm to roomtemperature. The reaction was quenched with water (10 ml) and aqueous10% NaHSO₄ (1 ml) and extracted with ethyl acetate (2×). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated invacuo. Chromatography (SiO₂, 10 to 3% methylene chloride/ethyl acetateeluent) gave 2.15 g (76%) of 2.

Step 2. Aqueous 1 N NaOH (3.5 ml, 3.5 mmol) was added to a solution of 2(340 mg, 0.74 mmol) in methanol and THF (6 ml of 1:1). The reaction wasstirred at room temperature for 18 hr. The reaction was evaporated,dissolved in water and acidified with aqueous 10% NaHSO₄ to pH 3. Theaqueous layer was extracted with ethyl acetate (3×) and the combinedorganic layers were dried anhydrous Na₂SO₄ and concentrated in vacuo togive 210 mg (64%) of 3.

Step 3. (3S) 3-(1-Fluorenylmethoxycarbonylamino)-4-oxobutyric acidtert-butyl ester semicarbazone (4; 226 mg, 0.5 mmol; prepared in asimilar manner as the benzyloxycarbonyl analog described in Graybill etal. Int. J. Protein Res., 44, pp. 173-82 (1994)) was dissolved in 10 mlof acetonitrile (20 ml) and diethylamine (2 ml) was added to thesolution. The reaction was stirred for two hours, concentrated in vacuo,the resulting dissolved in acetonitrile and concentrated in vacuo againto give (3S) 3-amino-4-oxobutyric acid tert-butyl ester semicarbazone. A5° C. solution of the semicarbazone and 3 (188 mg, 0.424 mmol) inmethylene chloride/DMF (6 ml of 1:1) was treated with1-hydroxybenzotriazole (HOBt; 57 mg, 0.424 mmol) and1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC; 115mg, 0.6 mmol) and the reaction was stirred at room temperature for 16hr. The reaction was diluted with ethyl acetate (100 ml) and washed withwater, aqueous saturated NaHCO₃ and aqueous saturated NaCl, dried overdried over anhydrous Na₂SO₄ and concentrated in vacuo. Chromatography(SiO₂, 5% ammonium hydroxide/5% methanol/methylene chloride eluent) gave250 mg of 5.

Step 4. Semicarbazone 5 (250 mg) was dissolved in 25% TFA/methylenechloride (10 ml) and stirred at room temperature for 5 hr. to give ayellow foam that was dissolved in 5 ml MeOH, 1 ml acetic Acid, and 1 mlof 37% aqueous formaldehyde that was stirred at room temperature for 18hrs. The reaction was concentrated in vacuo and the resulting gumpurified by chromatography (SiO₂, 1% formic acid/2% methanol/methylenechloride eluent) to afford 69 mg (30%) of Example 1 from compound 3.¹H-NMR (CD₃OD) δ2.42-2.54 (m, 1H); 2.60-2.76 (m, 1H); 4.22-4.38 (m, 1H);4.39-4.48 (m, 0.5H); 4.5-4.75 (m, 2.5H); 5.15 (s, 2H); 5.32 (br. s, 1H);7.2-7.86 (m, 14H).

EXAMPLE 2

Further data for Example 2 is found in Table 1.

(3S)-3-[3(R,S)-1,3-Dihydro-2-oxo-5-phenyl-((3,5-dichloro-4-methoxybenzoyl)amino)-2H-1,4-benzodiazepin-1-acetylamino]-4-oxobutyricacid

Step 1. MBHA resin (0.63 mmol/g, 4.14 g, 2.61 mmol) was suspended indimethylacetamide (20 mL) followed by addition of 6 (2.37 g, 4.0 mmol,prepared from (3S) 3-(fluorenylmethyloxycarbonyl)-4-oxobutryic acidt-butyl ester according to A. M. Murphy et. al. J. Am. Chem. Soc., 114,3156-3157 (1992)), O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU; 1.53 g, 4.04 mmol), and DIEA (1.75 mL, 10.0mmol). The reaction mixture was agitated 3 hr at room temperature usinga wrist arm shaker. The resin was isolated on a sintered glass funnel bysuction filtration and washed with dimethylacetamide (6×20 mL).Unreacted amine groups were then capped by reacting the resin with 20%(v/v) acetic anhydride/dimethylformamide (2×25 ml) directly in thefunnel (10 min wash). The resin was washed with dimethylformamide (3×50ml), dichloromethane (3×50 ml) and methanol (3×20 mL) prior to dryingovernight in vacuo to yield 7 (5.33 g, 0.35 mmol/g, 1.86 mmol, 71%).

Step 2. Resin 7 (4.0 g, 0.35 mmol/g, 1.4 mmol) was swelled in a sinteredglass funnel by washing with dimethylformamide (3×25 mL). The Fmocprotecting group was then cleaved with 25% (v/v)piperidine/dimethylformamide (25 mL) for 10 min (intermittent stirring)and then for 20 min with fresh piperidine reagent (25 ml). The resin wasthen washed with dimethylformamide (3×25 ml), followed byN-methypyrrolidone (2×25 mL). After transferring the resin to a 100 mLflask, N-methypyrrolidone was added to obtain a slurry followed by 8(0.958 g, 2.1 mmol), HOBT-H₂O (0.321 g, 2.1 mmol), HBTU (0.796 g, 2.1mmol) and DIEA (0.732 mL, 4.2 mmol). The reaction mixture was agitatedovernight at room temperature using a wrist arm shaker. The resinwork-up was performed as described for 7 to yield 9 (4.17 g, 0.27mmol/g, 1.12 mmol, 80%).

Step 3. This compound was prepared from resin 9 (0.17 g, 0.047 mmol)using an Advanced ChemTech 396 Multiple Peptide synthesizer. Theautomated cycles consisted of a resin wash with dimethylformamide (3×1mL), deprotection with 25% (v/v) piperidine in dimethylformamide (1 mL)for 3 min followed by fresh reagent (1 mL) for 10 min. The resin 10 waswashed with dimethylformamide (3×1 mL) and N-methypyrrolidone (3×1 mL).

Step 4. Resin 10 was acylated with a solution of 0.4M3,5-dichloro-4-methoxybenzoic acid and 0.4M HOBT in N-methypyrrolidone(0.5 mL), a solution of 0.4M HBTU in N-methylpyrrolidone (0.5 mL) and asolution of 1.6M DIEA in N-methypyrrolidone (0.25 mL) and the reactionwas shaken for 2 hr at room temperature. The acylation step wasrepeated. Finally, the resin was washed with dimethylformamide (3×1 mL),dichloromethane (3×1 mL) and dried in vacuo yield resin 11.

Step 5. The aldehyde was cleaved from the resin 11 and globallydeprotected by treatment with 95% TFA/5% H₂O (v/v, 1.5 mL) for 30 min atroom temperature. After washing the resin with cleavage reagent (1 mL),the combined filtrates were concentrated to dryness in a Savant AES2000SpeedVac. The resulting pellets were dissolved in 50% acetonitrile/50%H₂O/0.1% TFA (5 mL) and lyophilized to obtain the crude product as anoff-white solid. The compound was purified by semi-prep RP-HPLC with aWaters DeltaPak C8 300A column (15μ, 30×300 mm) eluting with a linearacetonitrile gradient (20%-70%) containing 0.1% TFA (v/v) over 45 min at22 mL/min. Fractions containing the desired product were pooled andlyophilized to provide Example 2 (14.1 mg, 23.1 μmol, 49%).

EXAMPLE 3

Further data for Example 3 is found in Table 1.

(3S)-3-[3(R,S)-1,3-Dihydro-2-oxo-5-phenyl(benzoylamino)-2H-1,4-benzodiazepin-1-acetylamino]-4-oxobutyricacid

Step 4. Following a similar procedure as method 1, resin 5 was acylatedwith 0.5M benzoyl chloride in N-methypyrrolidone (1 mL) and 1.6M DIEA inN-methypyrrolidone (0.35 mL) for 3 hr at room temperature. The acylationstep was repeated to yield resin 12. This same methodology was alsoapplied to the preparation of sulfonylamino compounds by replacingbenzoyl chloride with sulfonyl chlorides. This same methodology was alsoapplied to the preparation of urea compounds by replacing benzoylchloride with the appropriate isocyanate.

Step 5. Cleavage of the aldehyde from the resin 12 and workup gaveExample 3 (31.6 mg).

EXAMPLES 4-27

Examples 4-27 were prepared by methods similar to the methods used toprepare Examples 2 or 3 (see Table 1).

TABLE 1 Ex. Structure  2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

23

24

25

26

27

HPLC RT (min) Prep. Ex. MF MW Purity (%) MS Example  2 C29H24Cl2N4O7611.44 13.8 88% (M + H) + 2 617.2  3 C28H24N4O6 512.53 10.43 96% (M +H) + 3 513.9  4 C30H27N5O7 569.58 8.93 98% (M + H) + 2 570.1  5C30H26ClN5O7 604.02 9.96 98% (M + H) + 2 605.4  6 C31H30N4O7 570.6112.25 94% (M + H) + 2 572.3  7 C28H23Cl2N5O6 596.43 11.9 98% (M + H) + 2597.8  8 C29H26N4O7 542.55 10.6 94% (M + H) + 2 543.9  9 C30H29N5O6555.60 9.35 93% (M + H) + 2 556.5 10 C29H26N4O7 542.55 10.78 94% (M +H) + 2 544.2 11 C31H25N5O6 563.57 11.8 96% (M + H) + 2 565.1 12C29H24N4O8 556.54 10.66 97% (M + H) + 2 558.1 13 C29H26N4O6 526.55 10.3694% (M + H) + 3 528.1 14 C28H30N4O6 518.57 10.75 94% (M + Na) + 3 541.915 C30H28N4O6 540.58 11.22 98% (M + H) + 3 541.6 16 C28H25N5O6 527.5410.24 97% (M + H) + 3 529.0 17 C29H27N5O6 541.57 9.60 98% (M + H) + 3542.7 18 C28H24ClN5O6 561.99 10.50 95% (M + H) + 2 562.7 19 C30H28N4O7556.58 10.42 98% (M + H) + 2 557.5 20 C28H24N4O7 528.53 9.00 98% (M +H) + 2 529.4 21 C20H22Cl2N4O7 597.42 11.55 98% (M + H) + 2 598.7 23C35H24Cl4N4O9 786.41 16.07 97% (M + H) + 2 787.2 24 C27H24N4O7S 548.5811.02 98% (M + H) + 3 549.9 25 C29H24N4O7 540.54 11.63 98% (M + H) + 2542.0 26 C32H26N4O6 562.59 12.23 98% (M + H) + 2 563.8 27 C32H26N4O6562.59 12.88 98% (M + H) + 3 564.1

EXAMPLE 28 ICE Inhibition

We obtained inhibition constants (K_(i)) and IC₅₀ for compounds of thisinvention using the three described below (Examples 28 and 31). Table 2this data for Examples 1-20 and 21-27.

TABLE 2 UV-Visible Cell PBMC Whole human blood Example Ki (nN) IC50 (nM)IC50 (nM) 1 650 20000 2 250 3 1500 4 525 5 300 6 180 7 30 >20000 8 500 9150 10 160 11 140 12 600 13 1000 14 7500 15 4000 16 4000 17 4000 1855 >20000 19 20 10000 20 155 21 2.5 10000 23 90 24 300 25 250 26 100 2745

1. Enzyme Assay with UV-visible Substrate

This assay is run using an Succinyl-Tyr-Val-Ala-Asp-p-Nitroanilidesubstrate. Synthesis of analogous substrates is described by L. A.Reiter (Int. J. Peptide Protein Res., 43, pp. 87-96 (1994)). The assaymixture contains:

 65 μl buffer (10 mM Tris, 1 mM DTT, 0.1% CHAPS @ pH 8.1)  10 μl ICE (50nM final concentration to give a rate of ˜1 mOD/min)  5 μlDMSO/Inhibitor mixture  20 μl 400 μM Substrate (80 μM finalconcentration) 100 μl total reaction volume

The visible ICE assay is run in a 96-well microtiter plate. Buffer, ICEand DMSO (if inhibitor is present) are added to the wells in the orderlisted. The components are left to incubate at room temperature for 15minutes starting at the time that all components are present in allwells. The microtiter plate reader is set to incubate at 37° C. Afterthe 15 minute incubation, substrate is added directly to the wells andthe reaction is monitored by following the release of the chromophore(pNA) at 405-603 mn at 37° C. for 20 minutes. A linear fit of the datais performed and the rate is calculated in mOD/min. DMSO is only presentduring experiments involving inhibitors, buffer is used to make up thevolume to 100 μl in the other experiments.

2. Enzyme Assay with Fluorescent Substrate

This assay is run essentially according to Thornberry et al. Nature 356pp. 768-774 (1992), using substrate 17 referenced in that article. Thesubstrate is: Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC). Thefollowing components are mixed:

 65 μl buffer (10 mM Tris, 1 mM DTT, 0.1% CHAPS @ pH 8.1)  10 μl ICE(2-10 nM final concentration)  5 μl DMSO/Inhibitor solution  20 μl 150μM Substrate (30 μM final) 100 μl total reaction volume

The assay is run in a 96-well microtiter plate. Buffer and ICE are addedto the wells. The components are left to incubate at 37° C. for 15minutes in a temperature-controlled wellplate. After the 15 minuteincubation, the reaction is started by adding substrate directly to thewells and the reaction is monitored at 37° C. for 30 minutes byfollowing the release of the AMC fluorophore using an excitationwavelength for 380 nm and an emission wavelength of 460 nm. A linear fitof the data for each well is performed and a rate is determined influorescence units per second.

For determination of enzyme inhibition constants (K_(i)) or the mode ofinhibition (competitive, uncompetitive or noncompetitive), the rate datadetermined in the enzyme assays at varying inhibitor concentrations arecomputer-fit to standard enzyme kinetic equations (see I. H. Segel,Enzyme Kinetics, Wiley-Interscience, 1975).

The determination of second order rate constants for irreversibleinhibitors was performed by fitting the fluorescence vs time data to theprogress equations of Morrison. Morrison, J. F., Mol. Cell. Biophys., 2,pp. 347-368 (1985). Thornberry et al. published a description of thesemethods for measurement of rate constants of irreversible inhibitors ofICE. Thornberry, N. A., et al. Biochemistry, 33, pp. 3923-3940 (1994).For compounds where no prior complex formation can be observedkinetically, the second order rate constants (k_(inact)) are deriveddirectly from the slope of the linear plots of k_(obs) vs. inhibitorconcentration [I]. For compounds where prior complex formation to theenzyme can be detected, the hyperbolic plots of k_(obs) vs. [I] are fitto the equation for saturation kinetics to first generate K_(i) and k′.The second order rate constant k_(inact) is then given by k′/K_(i).

3. PBMC Cell Assay

IL-L1β Assay with a Mixed Population of Human Peripheral BloodMononuclear Cells (PBMC) or Enriched Adherent Mononuclear Cells

Processing of pre-IL-1β by ICE can be measured in cell culture using avariety of cell sources. Human PBMC obtained from healthy donorsprovides a mixed population of lymphocyte subtypes and mononuclear cellsthat produce a spectrum of interleukins and cytokines in response tomany classes of physiological stimulators. Adherent mononuclear cellsfrom PBMC provides an enriched source of normal monocytes for selectivestudies of cytokine production by activated cells.

Experimental Procedure

An initial dilution series of test compound in DMSO or ethanol isprepared, with a subsequent dilution into RPMI-10% FBS media (containing2 mM L-glutamine, 10 mM HEPES, 50 U and 50 ug/ml pen/strep) respectivelyto yield drugs at 4× the final test concentration containing 0.4% DMSOor 0.4% ethanol. The final concentration of DMSO is 0.1% for all drugdilutions. A concentration titration which brackets the apparent K_(i)for a test compound determined in an ICE inhibition assay is generallyused for the primary compound screen.

Generally 5-6 compound dilutions are tested and the cellular componentof the assay is performed in duplicate, with duplicate ELISAdeterminations on each cell culture supernatant.

PBMC Isolation and IL-1 Assay

Buffy coat cells isolated from one pint human blood (yielding 40-45 mlfinal volume plasma plus cells) are diluted with media to 80 ml andLeukoPREP separation tubes (Becton Dickinson) are each overlaid with 10ml of cell suspension. After 15 min centrifugation at 1500-1800 xg, theplasma/media layer is aspirated and then the mononuclear cell layer iscollected with a Pasteur pipette and transferred to a 15 ml conicalcentrifuge tube (Corning). Media is added to bring the volume to 15 ml,gently mix the cells by inversion and centrifuge at 300 xg for 15 min.The PBMC pellet is resuspended in a small volume of media, the cells arecounted and adjusted to 6×10⁶ cells/ml.

For the cellular assay, 1.0 ml of the cell suspension is added to eachwell of a 24-well flat bottom tissue culture plate (Corning), 0.5 mltest compound dilution and 0.5 ml LPS solution (Sigma #L-3012; 20 ng/mlsolution prepared in complete RPMI media; final LPS concentration 5ng/ml). The 0.5 ml additions of test compound and LPS are usuallysufficient to mix the contents of the wells. Three control mixtures arerun per experiment, with either LPS alone, solvent vehicle control,and/or additional media to adjust the final culture volume to 2.0 ml.The cell cultures are incubated for 16-18 hr at 37° C. in the presenceof 5% CO₂.

At the end of the incubation period, cells are harvested and transferredto 15 ml conical centrifuge tubes. After centrifugation for 10 min at200 xg, supernatants are harvested and transferred to 1.5 ml Eppendorftubes. It may be noted that the cell pellet may be utilized for abiochemical evaluation of pre-IL-1β and/or mature IL-1β content incytosol extracts by Western blotting or ELISA with pre-IL-1β specificantisera.

Isolation of Adherent Mononuclear Cells

PBMC are isolated and prepared as described above. Media (1.0 ml) isfirst added to wells followed by 0.5 ml of the PBMC suspension. After aone hour incubation, plates are gently shaken and nonadherent cellsaspirated from each well. Wells are then gently washed three times with1.0 ml of media and final resuspended in 1.0 ml media. The enrichmentfor adherent cells generally yields 2.5-3.0×10⁵ cells per well. Theaddition of test compounds, LPS, cell incubation conditions andprocessing of supernatants proceeds as described above.

ELISA:

We have used Quantikine kits (R&D Systems) for measurement of matureIL-1β. Assays are performed according to the manufacturer's directions.Mature IL-1β levels of about 1-3 ng/ml in both PBMC and adherentmononuclear cell positive controls are observed. ELISA assays areperformed on 1:5, 1:10 and 1:20 dilutions of supernatants fromLPS-positive controls to select the optimal dilution for supernatants inthe test panel.

The inhibitory potency of the compounds can be represented by an IC₅₀value, which is the concentration of inhibitor at which 50% of matureIL-1β is detected in the supernatant as compared to the positivecontrols.

The skilled practitioner realizes that values obtained in cell assays,such as those described herein, can depend on multiple factors. Thevalues may not necessarily represent fine quantitative results.

EXAMPLE 29 Pharmacokinetic Studies in the Mouse

Peptidyl ICE inhibitors are cleared rapidly with clearance rates greaterthan 100 μ/min/kg. Compounds with lower clearance rates have improvedpharmacokinetic properties relative to peptidyl ICE inhibitors.

Clearance rates for compounds of this invention (μ/min/kg) may beobtained using the method described below:

Sample Preparation and Dosing

Compounds are dissolved in sterile TRIS solution (0.02M or 0.05M) at aconcentration of 2.5 mg/ml. Where necessary to ensure a completesolution, the sample is first dissolved in a minimum ofdimethylacetamide (maximum of 5% of total solution volume) then dilutedwith the TRIS solution.

The drug solution is administered to CD-1 mice (Charles RiverLaboratories—26-31 g) via the tail vein at a dose volume of 10 ml/kggiving a drug dose of 25 mg/kg, for example.

Mice may be dosed in groups (of 5, for example) for each timepoint(generally from 2 minutes to 2 hours) and then at the appropriate timethe animals are anaesthetised with halothane and the blood collectedinto individual heparinized tubes by jugular severance. The bloodsamples are cooled to 0° C. then the plasma separated and stored at −20°C. until assayed.

Bioassay

Drug concentration in the plasma samples are determined by HPLC analysiswith UV or MS (ESP) detection. Reverse phase chromatography is employedusing a variety of bonded phases from C1 to C18 with eluents composed ofaqueous buffer/acetonitrile mixtures run under isocratic conditions.

Quantitation is by external standard methods with calibration curvesconstructed by spiking plasma with drug solutions to give concentrationsin the range of 0.5 to 50 μg/ml.

Prior to analysis the plasma samples are deproteinated by the additionof acetonitrile, methanol, trichloroacetic acid or perchloric acidfollowed by centrifugation at 10,000 g for 10 minutes. Sample volumes of20 μl to 50 μl are injected for analysis.

Representative Dosing and Sampling Procedure

The drug is dissolved in sterile 0.02M Tris to give a 2.5 mg/ml solutionwhich is administered to 11 groups of 5 male CD-1 mice via the tail veinat a dose of 25 mg/kg. At each of the following timepoints: 2, 5, 10,15, 20, 30, 45, 60, 90 and 120 minutes a group of animals isanaesthetised and the blood collected into heparinized tubes. Afterseparation the plasma is stored at −20° C. until assayed.

Representative Assay

Aliquots of plasma (150 μl) are treated with 5% perchloric acid (5 μl)then mixed by vortexing and allowing to stand for 90 minutes prior tocentrifugation. The resulting supernatant is separated and 20 μl isinjected for HPLC analysis.

Representative HPLC Conditions

Column 100 × 4.6 mm Kromasil KR 100 5C4 Mobile Phase 0.1 m Tris pH 7.586% Acetonitrile 14% Flowrate 1 ml/min Detection UV at 210 nm RetentionTime 3.4 mins

EXAMPLE 30

Peptidyl ICE inhibitors are cleared rapidly with clearance rates greaterthan 80 ml/min/kg. Compounds with lower clearance rates have improvedpharmacokinetic properties relative to peptidyl ICE inhibitors.

The rate of clearance in the rat (ml/min/kg) for compounds of thisinvention may be obtained using the method described below:

In Vivo Rat Clearance Assay

Representative Procedure

Cannulations of the jugular and carotid vessels of rats under anesthesiaare performed one day prior to the pharmacokinetic study. M. J. Free, R.A. Jaffee; ‘Cannulation techniques for the collection blood and otherbodily fluids’; in: Animal Models; p. 480-495; N. J. Alexander, Ed.;Academic Press; (1978). Drug (10 mg/mL) is administered via the jugularvein in a vehicle usually consisting of: propylene glycol/saline,containing 100 mM sodium bicarbonate in a 1:1 ratio. Animals are dosedwith 10-20 mg drug/kg and blood samples are drawn at 0, 2, 5, 7, 10, 15,20, 30, 60, and 90 minutes from an indwelling carotid catheter. Theblood is centrifuged to plasma and stored at −20° C. until analysis.Pharmacokinetic analysis of data is performed by non-linear regressionusing standard software such as RStrip (MicroMath Software, UT) and/orPcnonlin (SCI Software, NC) to obtain clearance values.

Representative Analytical:

Rat plasma is extracted with an equal volume of acetonitrile (containing0.1% TFA). Samples are then centrifuged at approximately 1,000×g and thesupernatant analyzed by gradient HPLC. A typical assay procedure isdescribed below.

200 μL of plasma is precipitated with 200 μL of 0.1% trifluoroaceticacid (TFA) in acetonitrile and 10 μL of a 50% aqueous zinc chloridesolution, vortexed then centrifuged at ˜1000×g and the supernatantcollected and analyzed by HPLC.

HPLC procedure Column Zorbax SB-CN (4.6 × 150 mm) (5 μ particle size)Column temperature 50° C. Flow rate 1.0 mL/min Injection volume 75 μL.Mobile phase A = 0.1% TFA in water and B = 100% acetonitrile Gradientemployed 100% A to 30% A in 15.5 min  0% A at 16 min 100% A at 19.2 minWavelength 214 nm

A standard curve is run at 20, 10, 5, 2 and 1 μg/mL concentrations.

EXAMPLE 31 Whole Blood Assay for IL-1β Production

Whole blood assay IC₅₀ values for compounds of this invention areobtained using the method described below:

Purpose:

The whole blood assay is a simple method for measuring the production ofIL-1β (or other cytokines) and the activity of potential inhibitors. Thecomplexity of this assay system, with its full complement of lymphoidand inflammatory cell types, spectrum of plasma proteins and red bloodcells is an ideal in vitro representation of human in vivo physiologicconditions.

Materials:

Pyrogen-free syringes (˜30 cc)

Pyrogen-free sterile vacuum tubes containing lyophilized

Na₂EDTA (4.5 mg/10 ml tube)

Human whole blood sample (˜30-50 cc)

1.5 ml eppendorf tubes

Test compound stock solutions (˜25 mM in DMSO or other solvent)

Endotoxin -free sodium chloride solution (0.9%) and HBSS

Lipopolysaccharide (Sigma; Cat.# L-3012) stock solution at 1 mg/ml inHBSS

IL-1β ELISA Kit (R & D Systems; Cat # DLB50)

TNFα ELISA Kit (R & D Systems; Cat # DTA50)

Water bath or incubator

Whole Blood Assay Experimental Procedure:

Set incubator or water bath at 30° C. Aliquot 0.25 ml of blood into 1.5ml eppendorf tubes, making sure to invert the whole blood sample tubesafter every two aliquots. Differences in replicates may result if thecells sediment and are not uniformly suspended. Use of a positivedisplacement pipette will also minimize differences between replicatealiquots.

Prepare drug dilutions in sterile pyrogen-free saline by serialdilution. A dilution series which brackets the apparent K_(i) for a testcompound determined in an ICE inhibition assay is generally used for theprimary compound screen. For extremely hydrophobic compounds, preparecompound dilutions in fresh plasma obtained from the same blood donor orin PBS-containing 5% DMSO to enhance solubility.

Add 25 μl test compound dilution or vehicle control and gently mix thesample. Then add 5.0 μl LPS solution (250 ng/ml stocked prepared fresh:5.0 ng/ml final concentration LPS), and mix again. Incubate the tubes at30° C. in a water bath for 16-18 hr with occasional mixing.Alternatively, the tubes can be placed in a rotator set at 4 rpm for thesame incubation period. This assay should be set up in duplicate ortriplicate with the following controls: negative control—no LPS;positive control—no test inhibitor; vehicle control—the highestconcentration of DMSO or compound solvent used in the experiment.Additional saline is added to all control tubes to normalize volumes forboth control and experimental whole blood test samples.

After the incubation period, whole blood samples are centrifuged for 10minutes at ˜2000 rpm in the microfuge, plasma is transferred to a freshmicrofuge tube and centrifuged at 1000×g to pellet residual platelets ifnecessary. Plasma samples may be stored frozen at −70° C. prior to assayfor cytokine levels by ELISA.

ELISA:

R & D Systems (614 McKinley Place N.E. Minneapolis, Minn. 55413)Quantikine kits may be used for measurement of IL-1β and TNF-α. Theassays are performed according to the manufacturer's directions. IL-1βlevels of ˜1-5 ng/ml in positive controls among a range of individualsmay be observed. A 1:200 dilution of plasma for all samples is usuallysufficient for experiments for ELISA results to fall on the linear rangeof the ELISA standard curves. It may be necessary to optimize standarddilutions if you observe differences in the whole blood assay. Nerad, J.L. et al., J. Leukocyte Biol., 52, pp. 687-692 (1992).

EXAMPLE 32 Inhibition of ICE Homologs

1. Isolation of ICE Homologs Expression of TX in Insect Cells Using aBaculovirus Expression System.

Tx cDNA (C. Faucheu et al., EMBO, 14, p. 1914 (1995)) is subcloned intoa modified pVL1393 transfer vector, co-transfected the resultant plasmid(pVL1393/TX) into insect cells with viral DNA and the recombinantbaculovirus is identified. After the generation of high titerrecombinant virus stock, the medium is examined for TX activity usingthe visible ICE assay. Typically, infection of Spodoptera frugiperda(Sf9) insect cells at an MOI of 5 with recombinant virus stock result ina maximum expression after 48 hours of 4.7 μg/ml. ICE is used as astandard in the assay.

Amino terminal T7 tagged versions of ICE or TX are also expressed.Designed originally to assist the identification and purification of therecombinant proteins, the various constructs also allow examination ofdifferent levels of expression and of the relative levels of apoptosisexperienced by the different homologs. Apoptosis in the infected Sf9cells (examined using a Trypan Blue exclusion assay) is increased in thelines expressing ICE or TX relative to cells infected with the viral DNAalone.

Expression and Purification of N-terminally (His)₆-tagged CPP32 in E.coli.

A cDNA encoding a CPP32 (Fernanes-Alnemri et al., supra, 1994)polypeptide starting at Ser (29) is PCR amplified with primers that addin frame XhoI sites to both the 5′ and 3′ ends of the cDNA and theresulting XhoI fragment ligated into a Xho I-cut pET-15b expressionvector to create an in frame fusion with (his)₆ tag at the N-terminus ofthe fusion protein. The predicted recombinant protein starts with theamino acid sequence of MGSSHHHHHHSSGLVPRGSHMLE, where LVPRGS representsa thrombin cleavage site, followed by CPP32 starting at Ser (29). E.coli BL21(DE3) carrying the plasmid are grown to log phase at 30° C. andare then induced with 0.8 mM IPTG. Cells are harvested two hours afterIPTG addition. Lysates are prepared and soluble proteins are purified byNi-agarose chromatography. All of the expressed CPP32 protein would bein the processed form. N-terminal sequencing analysis should indicatethat the processing has occurred at the authentic site between Asp (175)and Ser (176). Approximately 50 μg of CPP32 protein from 200 ml culturecould be obtained. As determined by active site titration, the purifiedproteins are fully active. The protease preparations are also veryactive in vitro in cleaving PARP as well as the synthetic DEVD-AMCsubstrate (Nicholson et al., 1995).

2. Inhibition of ICE Homologs

The selectivity of a panel of reversible inhibitors for ICE homologs maybe obtained. ICE enzyme assays are performed according to Wilson et al.(1994) using a YVAD-AMC substrate (Thornberry et al., 1992). Assay of TXactivity is performed using the ICE substrate under identical conditionsto ICE. Assay of CPP32 is performed using a DEVD-AMC substrate(Nicholson et al., 1995).

Second-order rate constants for inactivation of ICE and ICE homologswith irreversible inhibitors are obtained.

EXAMPLE 33 Inhibition of Apoptosis

Fas-Induced Apoptosis in U937 Cells

Compounds may be evaluated for their ability to block anti-Fas-inducedapopotosis. Using RT-PCR, mRNA encoding ICE, TX, ICH-1, CPP32 and CMH-1in unstimulated U937 cells may be detected. This cell line may be usedfor apoptosis studies. For example, U937 cells are seeded in culture at1×10⁵ cells/ml and grown to ˜5×10⁶ cells/ml. For apoptosis experiments,2×10⁶ cells are plated in 24-well tissue culture plates in 1 mlRPMI-1640-10% FBS and stimulated with 100 ng/ml anti-Fas antigenantibody (Medical and Biological Laboratories, Ltd.). After a 24 hrincubation at 37° C., the percentage of apoptotic cells is determined byFACS analysis using ApoTag reagents.

All compounds are tested initially at 20 μM and titrations are performedwith active compounds to determine IC₅₀ values.

EXAMPLE 34 In Vivo Acute Assay for Efficacy as Anti-inflammatory Agent

LPS-Induced IL-1β Production

Efficacy is evaluated in CD1 mice (n=6 per condition, for example)challenged with LPS (20 mg/kg IP). The test compounds are prepared inolive oil:DMSO:ethanol (90:5:5) and administered by IP injection onehour after LPS. Blood is collected seven hours after LPS challenge.Serum IL-1β levels are measured by ELISA.

Compounds may also be administered by oral gavage to assess absorption.Compounds administered orally that inhibit IL-1β secretion aresuggestive of the potential oral efficacy of those compounds as ICEinhibitors and thus as anti-inflammatory agents.

EXAMPLE 35 Measurement of Blood Levels of Prodrugs

Mice are administered a p.o. (oral) dose of compounds (50 mg/kg, forexample) prepared in 0.5% carboxymethylcellulose. Blood samples arecollected at 1 and 7 hours after dosing. Serum is extracted byprecipitation with an equal volume of acetonitrile containing 2% formicacid followed by centrifugation. The supernatant is analyzed by liquidchromatography-mass spectrometry (ESI-MS) with a detection level of 0.03to 3 μg/ml. Detectable blood levels are thus determined.

EXAMPLE 36 ICE Inhibition Assays—IGIF

IGIF may be substituted for IL-1 in the ICE inhibition assays describedin Example 28. Thus, the ability of ICE inhibitors to decrease IGIFproduction may be determined.

For example, to run the human PBMC assay, human buffy coat cells may beobtained from blood donors and peripheral blood mononuclear cells (PBMC)isolated by centrifugation in LeukoPrep tubes (Becton-Dickinson, LincolnPark, N.J.). PBMC are added (3×10⁶/well) to 24 well Corning tissueculture plates and after 1 hr incubation at 37° C., non-adherent cellsare removed by gently washing. Adherent mononuclear cells are stimulatedwith LPS (1 μg/ml) with or without ICE inhibitor in 2 ml RPMI-1640-10%FBS. After 16-18 hr incubation at 37° C., IGIF and IFN- are quantitatedin culture supernatants by ELISA.

EXAMPLE 37

The antiviral efficacy of compounds may be evaluated in various in vitroand in vivo assays. For example, compounds may be tested in in vitroviral replication assays. In vitro assays may employ whole cells orisolated cellular components. In vivo assays include animal models forviral diseases. Examples of such animal models include, but are notlimited to, rodent models for HBV or HCV infection, the Woodchuck modelfor HBV infection, and chimpanzee model for HCV infection.

ICE inhibitors may also be evaluated in animal models for dietaryalcohol-induced disease.

EXAMPLES 38-59

Examples 38-56 (Table 3) were prepared by methods similar to the methodsused to prepare Examples 2 or 3. Examples 57-59 (Table 3) were preparedby methods similar to the methods used to prepare Example 1.

TABLE 3 Ex. Structure 38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

EXAMPLE 60 ICE Inhibition

We obtained inhibition constants (K_(i)) and IC₅₀ values for compoundsof this invention using the methods described herein (see Examples 28and 31). Table 4 lists this data for Examples 11, 38-56 and 58-59.

TABLE 4 UV-Visible Cell PBMC Whole human blood Example Ki (nN) IC50 (nM)IC50 (nM) 11 140 9000 38 590 39 260 >20000 40 100 41 350 42 1200 43 170044 2400 45 450 46 180 47 1500 48 2300 49 48 >8000 50 40 >8000 51 1000 521500 53 700 54 300 55 7500 56 135 58 48 >20000 59 60 9200

EXAMPLE 61 Mouse Carrageenan Peritoneal Inflammation RepresentativeProcedure

Inflammation is induced in mice with an intraperitoneal (IP) injectionof 10 mg carrageenan in 0.5 ml of saline (Griswold et al., Inflammation,13, pp. 727-739 (1989)). Drugs are administered by oral gavage inethanol/PEG/water, β-cyclodextrin, labrosol/water or cremophor/watervehicle. The mice are sacrificed at 4 hours post carrageenanadministration, then injected IP with 2 ml of saline containing 5 U/mlheparin. After gentle massage of the peritoneum, a small incision ismade, the contents collected and volume recorded Samples are kept on iceuntil centrifuged (130×g, 8 mins at 4° C.) to remove cellular material,and the resultant supernatant stored at −20° C. IL-1β levels in theperitoneal fluid are determined by ELISA.

EXAMPLE 62 Type II Collagen-induced Arthritis Representative Procedure

Type II collagen-induced arthritis is established in male DBA/1J mice atdescribed Wooley and Geiger (Wooley, P. H., Methods in Enzymology, 162,pp. 361-373 (1988) and Geiger, T., Clinical and ExperimentalRheumatology, 11, pp. 515-522 (1993)). Chick sternum Type II collagen (4mg/kg in 10 mM acetic acid) is emulsified with an equal volume ofFreund's complete adjuvant (FCA) by repeated passages (400) between two10 ml glass syringes with a gauge 16 double-hub needle. Mice areimmunized by intradermal injection (50 l; 100 l CII per mouse) ofcollagen emulsion 21 days later at the contra-lateral side of the tailbase. Drugs are administered twice a day (10, 25 and 50 mg/kg) by oralgavage approximately 7 h apart. Vehicles that may be used includeethanol/PEG/water, β-cyclodextrin, labrosol/water or cremophor/water.Drug treatments are initiated within 2 h of the CII boosterimmunization. Inflammation is scored on a 1 to 4 scale of increasingseverity on the two front paws and the scores are added to give thefinal score.

EXAMPLE 63 In Vivo Bioavailability Determination

Representative Procedure

The drugs (10-100 mg/kg) are dosed orally to rats (10 mL/kg) inethanol/PEG/water, β-cyclodextrin, labrosol/water or cremophor/water.Blood samples are drawn from the carotid artery at 0.25, 0.50, 1, 1.5,2, 3, 4, 6, and 8 hours after dosing, centrifuged to plasma and storedat −70° C. until analysis. Aldehyde concentrations are determined usingan enzymatic assay. Pharmacokinetic analysis of data is performed bynon-linear regression using RStrip (MicroMath Software, UT). Drugavailability values are determined as follows: (AUC of drug after oralprodrug dosing/AUC of drug after i.v. dosing of drug)×(dose i.v./dosep.o.)×100%.

The data of the examples above demonstrate that compounds according tothis invention display inhibitory activity towards IL-1β ConvertingEnzyme and that ICE controls IGIF and IFN-γ levels.

Insofar as the compounds of this invention are able to inhibit ICE invitro and furthermore, may be delivered orally to mammals, they are ofevident clinical utility for the treatment of IL-1-, apoptosis-, IGIF-,and IFN-γ-mediated diseases. These tests are predictive of the compoundsability to inhibit ICE in vivo.

While we have described a number of embodiments of this invention, it isapparent that our basic constructions may be altered to provide otherembodiments which utilize the products and processes of this invention.

What is claimed is:
 1. A compound represented by formula (III):

wherein: Y is

C is pyrido, wherein any hydrogen bound to any ring atom is optionallyreplaced by —R⁴, R¹ is -aryl, -heteroaryl, -alkylaryl, or-alkylheteroaryl, wherein each aryl or heteroaryl is optionally singlyor multiply substituted with R¹⁷; R² is a bond, —CH₂C(O)—, —C(O)—,—C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—, —N(H)S(O)₂—, —N(H)C(O)C(O)—,—CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—, —N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—,—N(R¹⁹)C(O)C(O)—, —N(R¹⁹)CH₂C(O)—, or —C(O)C(═NOR¹¹)—, provided thatwhen R² is not a bond, R² is bonded to the NH attached to the 7-memberedring through carbonyl or sulfonyl; R³ is -aryl, -heteroaryl,-cycloalkyl, -alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H) (alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or —CH₂N(alkyl)₂; R⁵ is—OH, —OR⁸, or —N(H)OH; R⁶ is —H, —CH₂OR⁹, —CH₂SR¹⁰, —CH₂NHR⁹,—CH₂N(R⁹)R¹², —C(H)N₂, —CH₂F, —CH₂Cl, —C(O)N(R¹¹)R¹², —R¹³, or —R¹⁴; R⁸is -alkyl, -cycloalkyl, -aryl, -heteroaryl, -alkylaryl,-alkylheteroaryl, or alkylheterocycle; R⁹ is —H, —C(O)aryl,—C(O)heteroaryl, —C(O)alkylaryl, —C(O)alkylheteroaryl, -alkylaryl,-alkylheteroaryl, -aryl, -heteroaryl, or —P(O)R¹⁵R¹⁶; R¹⁰ is -alkylaryl,-aryl, -heteroaryl, or -alkylheteroaryl; each R¹¹ and R¹² isindependently —H, -alkyl, -aryl, -heteroaryl, -cycloalkyl, -alkylaryl,or -alkylheteroaryl; R¹³ is -alkylaryl, -alkenylaryl, -alkynylaryl, or-alkylheteroaryl; R¹⁴ is

wherein any hydrogen bound to (i) is optionally replaced with R¹⁷ andany hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ or R²⁰;each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, -alkylheteroaryl, —Oalkyl, —Oaryl,—Oheteroaryl, —Oalkylaryl, or —Oalkylheteroaryl; R¹⁷ is —OH, —F, —Cl,—Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂, —N(H)C(O)H, —N(H)C(O)NH₂,—S(O)₂NH₂, —C(O)H, -alkyl, -cycloalkyl, -perfluoroalkyl, —O-alkyl,—N(H)alkyl, —N(alkyl)₂, —CO₂alkyl, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S(O)₂N(H)alkyl,—S(O)₂N(alkyl)₂, —S-alkyl, —S(O₂)alkyl, or —C(O)alkyl; R¹⁸ is -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl, —O-heteroaryl,—O-alkylaryl, —O-alkylheteroaryl, —N(H)aryl, —N(aryl)₂, —N(H)heteroaryl,—N(heteroaryl)₂, —N(H)alkylaryl, —N(alkylaryl)₂, —N(H)alkylheteroaryl,—N(alkylheteroaryl)₂, —S-aryl, —S-heteroaryl, —S-alkylaryl,—S-alkylheteroaryl, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl,—C(O)alkyheteroaryl, —CO₂aryl, —CO₂heteroaryl, —CO₂alkylaryl,—CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂, —C(O)N(H)heteroaryl,—C(O)N(heteroaryl)₂, —C(O)N(H)alkylaryl, —C(O)N(alkylaryl)₂,—C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂, —S(O)₂-aryl,—S(O)₂-heteroaryl, —S(O)₂-alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂N(H)aryl, —S(O₂)N(H)heteroaryl, —S(O₂)N(H)alkylaryl,—SO)₂N(H)alkylheteroaryl, —S(O)₂N(aryl)₂, —S(O)₂N(H)(heteroaryl)₂,—S(O)₂N(alkylaryl)₂, —S(O)₂N(alkylheteroaryl)₂, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroaryl, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂, or—N(H)C(O)N(alkylheteroaryl)₂; R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, or -alkylheterocycle; R²⁰ is-alkyl-R¹⁸; m is 0 or 1; and X is O or S.
 2. A compound represented byformula (IV),

wherein Y is:

C is pyrido, wherein any hydrogen bound to any ring atom is optionallyreplaced by —R⁴; R¹ is -aryl, -heteroaryl, -alkylaryl, or-alkylheteroaryl, wherein each aryl or heteroaryl is optionally singlyor multiply substituted with R¹⁷; R² is a bond, —CH₂C(O)—, —C(O)—,—C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—, —N(H)S(O)₂—, —N(H)C(O)C(O)—,—CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—, —N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—,—N(R¹⁹)C(O)C(O)—, —N(R¹⁹)CH₂C(O)—, or —C(O)C(═NOR¹¹)—, provided thatwhen R² is not a bond, R² is bonded to the NH attached to the 7-memberedring through carbonyl or sulfonyl; R³ is -aryl, -heteroaryl,-cycloalkyl, -alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H)(alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or —CH₂N(alkyl)₂; R⁶ is—H, —CH₂OR⁹, —CH₂SR¹⁰, —CH₂N(H)R⁹, —CH₂N(R⁹)R¹², —CHN₂, —CH₂F, —CH₂Cl,—C(O)N(R¹¹)R¹², —R¹³, or —R¹⁴; R⁷ is —C(O)alkyl, —C(O)cycloalkyl,—C(O)alkyenyl, —C(O)alkylaryl, —C(O)alkylheteroaryl, —C(O)heterocycle,or —C(O)alkylheterocycle; R⁸ is -alkyl, -cycloalkyl, -aryl, -heteroaryl,-alkylaryl, -alkylheteroaryl, or alkylheterocycle; R⁹ is —H, —C(O)aryl,—C(O)heteroaryl, —C(O)alkylaryl, —C(O)alkylheteroaryl, -alkylaryl,-alkylheteroaryl, or —P(O)R¹⁵R¹⁶; R¹⁰ is -alkylaryl or -alkylheteroaryl;each R¹¹ and R¹² is independently —H, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, or -alkylheteroaryl; R¹³ is -alkylaryl,-alkenylaryl, -alkynylaryl, or -alkylheteroaryl; R¹⁴ is

wherein any hydrogen bound to (i) is optionally replaced with R¹⁷ andany hydrogen bound to (ii) is optionally replaced with R¹⁷, R¹⁸ or R²⁰;each R¹⁵ and R¹⁶ is independently —H, —OH, -alkyl, -aryl, -heteroaryl,-cycloalkyl, -alkylaryl, -alkylheteroalkyl, —Oalkyl, —Oaryl,—Oheteroaryl, —Oalkylaryl, or —Oalkylheteroaryl; R¹⁷ is —OH, —F, —Cl,—Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂, —N(H)C(O)H, —N(H)C(O)NH₂,—S(O₂)NH₂, —C(O)H, -alkyl, -cycloalkyl, -perfluoroalkyl, —O-alkyl,—N(H)alkyl, —N(alkyl)₂, —CO₂alkyl, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S(O)₂N(H)alkyl,—S(O)₂N(alkyl)₂, —S-alkyl, —S(O)₂alkyl, or —C(O)alkyl; R¹⁸ is -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, —O-aryl, —O-heteroaryl,—O-alkylaryl, —O-alkylheteroaryl, —N(H)aryl, —N(aryl)₂, —N(H)heteroaryl,—N(heteroaryl)₂, —N(H)alkylaryl, —N(alkylaryl)₂, —N(H)alkylheteroaryl,—N(alkylheteroaryl)₂, —S-aryl, —S-heteroaryl, —S-alkylaryl,—S-alkylheteroaryl, —C(O)aryl, —C(O)heteroaryl, —C(O)alkylaryl,—C(O)alkyheteroaryl, —CO₂aryl, —CO₂heteroaryl, —CO₂alkylaryl,—CO₂alkylheteroaryl, —C(O)N(H)aryl, —C(O)N(aryl)₂, —C(O)N(H)heteroaryl,—C(O)N(heteroaryl)₂, —C(O)N(H)alkylaryl, —C(O)N(alkylaryl)₂,—C(O)N(H)alkylheteroaryl, —C(O)N(alkylheteroaryl)₂, —S(O)₂aryl,—S(O)₂heteroaryl, —S(O)₂alkylaryl, —S(O)₂alkylheteroaryl,—S(O)₂N(H)aryl, —S(O)₂N(H)heteroaryl, —S(O)₂N(H)alkylaryl,—S(O₂)N(H)alkylheteroaryl, —S(O)₂N(aryl)₂, —S(O₂)N(heteroaryl)₂,—S(O)₂N(alkylaryl)₂, —S(O)₂N(alkylheteroaryl)₂, —N(H)C(O)N(H)aryl,—N(H)C(O)N(H)heteroaryl, —N(H)C(O)N(H)alkylaryl,—N(H)C(O)N(H)alkylheteroaryl, —N(H)C(O)N(aryl)₂,—N(H)C(O)N(heteroaryl)₂, —N(H)C(O)N(alkylaryl)₂, or—N(H)C(O)N(alkylheteroaryl)₂; R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl,-heteroaryl, -alkylaryl, -alkylheteroaryl, or -alkylheterocycle; R²⁰ is-alkyl-R¹⁸; m is 0 or 1; and X is O or S.
 3. The compound according toclaims 1 or 2, wherein: C is pyrido, wherein no hydrogen is replaced. 4.The compound according to claims 1 or 2, wherein: Y is

C is pyrido, wherein any hydrogen bound to any ring atom is optionallyreplaced by R⁴; R¹ is -phenyl, -naphthyl, or -isoquinolinyl, eachoptionally singly or multiply substituted with R¹⁷, wherein R¹⁷ is —OH,—NH₂, —Cl, —F, —Oalkyl, or —N(alkyl)₂; R² is —C(O)—, —S(O)₂—,—C(O)C(O)—, or —CH₂C(O)—; R³ is -methyl, -ethyl, -n-propyl, -isopropyl,-phenyl, -2-pyridinyl, -3-pyridinyl, -4-pyridinyl, or -thiazolyl; R⁴ is-fluoro or -chloro; R⁵ is OH; R⁶ is —H or —R¹⁴; and X is O.
 5. Apharmaceutical composition comprising a compound according to any one ofclaims 1-4 and a pharmaceutically acceptable carrier, adjuvant orvehicle.
 6. A process for preparing a compound represented by formula(V):

wherein: R²¹ is:

C is pyrido, wherein any hydrogen bound to any ring atom is optionallyreplaced by R⁴; R²² is:

each R²³ is independently -alkyl, -cycloalkyl, -aryl, -heteroaryl,-alkylaryl, -alkylheteroaryl, or -alkylheterocycle; R¹ is -aryl,-heteroaryl, -alkylaryl, or -alkylheteroaryl, wherein each aryl orheteroaryl is optionally singly or multiply substituted with R¹⁷; R² isa bond, —CH₂C(O)—, —C(O)—, —C(O)C(O)—, —S(O)₂—, —OC(O)—, —N(H)C(O)—,—N(H)S(O)₂—, —N(H)C(O)C(O)—, —CH═CHC(O)—, —OCH₂C(O)—, —N(H)CH₂C(O)—,—N(R¹⁹)C(O)—, —N(R¹⁹)S(O)₂—, —N(R¹⁹)C(O)C(O)—, or —N(R¹⁹)CH₂C(O), or—C(O)C(═NOR¹¹)—, provided that when R² is not a bond, R² is bonded tothe NH attached to the 7-membered ring through carbonyl or sulfonyl; R³is -aryl, -heteroaryl, -cycloalkyl, -alkyl, —N(alkyl)₂,

R⁴ is —OH, —F, —Cl, —Br, —I, —NO₂, —CN, —NH₂, —CO₂H, —C(O)NH₂,—N(H)C(O)H, —N(H)C(O)NH₂, -alkyl, -cycloalkyl, -perfluoroalkyl,—O-alkyl, —N(H)(alkyl), —N(alkyl)₂, —C(O)N(H)alkyl, —C(O)N(alkyl)₂,—N(H)C(O)alkyl, —N(H)C(O)N(H)alkyl, —N(H)C(O)N(alkyl)₂, —S-alkyl,—S(O₂)alkyl, —C(O)alkyl, —CH₂NH₂, —CH₂N(H)alkyl, or —CH₂N(alkyl)₂; R₁₁is —H, -alkyl, -aryl, -heteroaryl, -cycloalkyl, -alkylaryl, or-alkylheteroaryl; R¹⁹ is —H, -alkyl, -cycloalkyl, -aryl, -heteroaryl,-alkylaryl, -alkylheteroaryl, or -alkylheterocycle; and m is 1 or 2;comprising the steps of: a) reacting a compound represented by formula(VI): R²¹—OH, wherein R²¹ is as defined above, with a compoundrepresented by formula (VII):

wherein R²³ is as defined above, in the presence of an inert solvent,triphenylphoshine, a nucleophilic scavenger, and tetrakis-triphenylphosphine palladium(0) at ambient temperature under an inert atmosphere;and b) adding to the mixture formed in step a), HOBT and EDC.
 7. Theprocess according to claim 6, wherein: C is pyrido, wherein no hydrogenis replaced.
 8. The process according to claim 6, wherein: C is pyrido,wherein any hydrogen bound to any ring atom is optionally replaced byR⁴; R¹ is phenyl, naphthyl, or isoquinolinyl, each optionally singly ormultiply substituted with R¹⁷, wherein R¹⁷ is —OH, —NH₂, —Cl, —F,—Oalkyl, or —N(alkyl)₂; R² is —C(O)—, —S(O)₂—, —C(O)C(O)—, or —CH₂C(O)—;R³ is methyl, ethyl, n-propyl, isopropyl, phenyl, or thiazolyl; R⁴ is-fluoro or -chloro; and m is
 1. 9. The process according to any one ofclaims 6-8, wherein the inert solvent is CH₂Cl₂, DMF, or a mixture ofCH₂Cl₂ and DMF.
 10. The process according to any one of claims 6-8,wherein the nucleophilic scavenger is dimedone, morpholine, or dimethylbarbituric acid.
 11. The process according to claim 10, wherein thenucleophilic scavenger is dimethyl barbituric acid.
 12. The processaccording to claim 10, wherein the inert solvent is CH₂Cl₂, DMF, or amixture of CH₂Cl₂ and DMF.
 13. The process according to claim 12,wherein the nucleophilic scavenger is dimethyl barbituric acid.
 14. Amethod for treating a disease selected from osteoarthritis,pancreatitis, asthma, adult respiratory distress syndrome,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Grave's disease, autoimmune gastritis,insulin-dependent diabetes mellitus (Type I), autoimmune hemolyticanemia, autoimmune neutropenia, thrombocytopenia, chronic activehepatitis, myasthenia gravis, inflammatory bowel disease, Crohn'sdisease, psoriasis, graft vs host disease, osteoporosis, multiplemyeloma-related bone disorder, acute myelogenous leukemia, chronicmyelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiplemyeloma, sepsis, septic shock, Shigellosis, cerebral ischemia,myocardial ischemia, spinal muscular atrophy, or neurological damage dueto stroke in a patient comprising the step of administering to saidpatient a pharmaceutical composition according to claim
 5. 15. Themethod according to claim 14, wherein the disease is osteoarthritis,acute pancreatitis, rheumatoid arthritis, inflammatory bowel disease,Crohn's disease, or psoriasis.